Polymerases engineered to reduce nucleotide-independent dna binding

ABSTRACT

Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By Binding™ procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.

RELATED APPLICATIONS

This application claims the benefit of U.S. patent application Ser. No.15/581,822, filed Apr. 28, 2017, which claims the benefit of U.S.Provisional Application No. 62/444,733, filed Jan. 10, 2017, and U.S.Provisional Application No. 62/329,489, filed Apr. 29, 2016; and U.S.Provisional Application No. 62/534,871, filed Jul. 20, 2017. Thedisclosures of these earlier applications are hereby incorporated byreference in their entireties.

TECHNICAL FIELD

The present disclosure generally relates to the field of biotechnology.More specifically, the disclosure relates to engineered DNA polymeraseshaving unique activity profiles, including reduced affinity for primedtemplate nucleic acids in the absence of cognate nucleotides.

BACKGROUND

Naturally occurring DNA polymerizing enzymes are responsible foraccurately replicating DNA within the cells of an organism. This processinvolves catalysis at the 3′-end of a growing DNA strand, whereby a freedeoxyribonucleotide triphosphate (dNTP) having a base moiety matched tothe base moiety on the complementary template strand is incorporated.This requirement for complementarity is utilized by sequencingtechnologies to analyze DNA for medical, industrial, and scientificapplications.

Indeed, DNA polymerases and fragments thereof are important tools fordetermining identity of the next correct nucleotide (i.e., the “cognate”nucleotide) of a template nucleic acid, whether for detection of singlenucleotide polymorphisms (SNPs) or more extensive sequencedetermination. Example applications include sequencing-by-synthesis,where cognate nucleotide identification follows nucleotideincorporation; and Sequencing By Binding™ technology, where cognatenucleotide identification is based on observations or measurements ofbinding events taking place prior to, or without, nucleotideincorporation.

Given the utility and advantages of Sequencing By Binding™ technology,there is an ongoing need for new and useful tools and methods that canbe used for enhancing discrimination between cognate and non-cognatenucleotide in the sequencing procedure. The present disclosure addressesthis need.

SUMMARY OF THE DISCLOSURE

In one aspect, the disclosure relates to an engineered DNA polymerasethat includes a variant of the sequence of SEQ ID NO:3, with the variantbeing at least 80% identical to SEQ ID NO:3 and including an amino acidsubstitution mutation at one or more of positions K250, Q281, D355,Q425, and D532. According to one generally preferred embodiment, thevariant is at least 90% identical to SEQ ID NO:3. Preferably, thevariant is at least 95% identical to SEQ ID NO:3. More preferably, thevariant is at least 98% identical to SEQ ID NO:3. According to someembodiments, when the variant is at least 90% identical to SEQ ID NO:3,the sequence of SEQ ID NO:5 can be joined to the amino terminus thereof.Alternatively, when the variant is at least 90% identical to SEQ IDNO:3, the sequence of SEQ ID NO:6 can be joined to the amino terminusthereof. According to another generally preferred embodiment, thesubstitution mutation at position K250 involves a mutation to a polaramino acid, the substitution mutation at position Q281 involves amutation to an acidic amino acid, the substitution mutation at positionD355 involves a mutation to a different acidic amino acid, thesubstitution mutation at position Q425 involves a mutation to adifferent polar amino acid, and the substitution mutation at positionD532 involves a mutation to a different acidic amino acid. Morepreferably, the substitution mutation at position K250 can involve amutation to Cys, the substitution mutation at position Q281 can involvea mutation to Glu, the substitution mutation at position D355 caninvolve a mutation to Glu, the substitution mutation at position Q425can involve a mutation to Cys, and the substitution mutation at positionD532 can involve a mutation to Glu. According to another generallypreferred embodiment, the variant involves replacement of up to 10 aminoacids of SEQ ID NO:3. Preferably, the variant includes replacement of upto 5 amino acids of SEQ ID NO:3. According to another generallypreferred embodiment, the mutant DNA polymerase is present in a ternarycomplex that further includes a primed template nucleic acid and acognate nucleotide or analog thereof. Preferably, the cognate nucleotideor analog thereof includes an exogenous fluorescent label. According toanother generally preferred embodiment, the at least one amino acidsubstitution mutation is a substitution mutation at position Q281 thatreplaces Gln (Q) with Glu (E). According to another generally preferredembodiment, the at least one amino acid substitution mutation is asubstitution mutation at position K250 that replaces Lys (K) with Cys(C), and a substitution mutation at position Q425 that replaces Gln (Q)with Cys (C). According to another generally preferred embodiment, theat least one amino acid substitution mutation is a substitution mutationat position Q281 that replaces Gln (Q) with Glu (E), a substitutionmutation at position K250 that replaces Lys (K) with Cys (C), and asubstitution mutation at position Q425 that replaces Gln (Q) with Cys(C). According to another generally preferred embodiment, the at leastone amino acid substitution mutation is a substitution mutation atposition D355 that replaces Asp (D) with Glu (E), and a substitutionmutation at position Q281 that replaces Gln (Q) with Glu (E). Accordingto another generally preferred embodiment, the at least one amino acidsubstitution mutation is a substitution mutation at position D355 thatreplaces Asp (D) with Glu (E), a substitution mutation at position K250that replaces Lys (K) with Cys (C), and a substitution mutation atposition Q425 that replaces Gln (Q) with Cys (C). According to anothergenerally preferred embodiment, the at least one amino acid substitutionmutation is a substitution mutation at position D355 that replaces Asp(D) with Glu (E), a substitution mutation at position Q281 that replacesGln (Q) with Glu (E), a substitution mutation at position K250 thatreplaces Lys (K) with Cys (C), and a substitution mutation at positionQ425 that replaces Gln (Q) with Cys (C). According to another generallypreferred embodiment, the engineered DNA polymerase further includes anexogenous label covalently joined thereto. Preferably, the exogenouslabel includes a fluorescent label. According to another generallypreferred embodiment, the engineered DNA polymerase includesMg²⁺-dependent phosphodiester bond forming activity. According toanother generally preferred embodiment, the differential affinity of theengineered DNA polymerase for the primed template nucleic acid in thepresence and absence of cognate nucleotide is greater than thedifferential affinity of the DNA polymerase of SEQ ID NO:4 for theprimed template nucleic acid in the presence and absence of cognatenucleotide.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Glu (E) at position 290. According to one generallypreferred embodiment, the mutant DNA polymerase further includes anN-terminal polypeptide sequence appended to the sequence of SEQ ID NO:2.Preferably, the variant sequence is a variant of SEQ ID NO:1. Morepreferably, the mutant DNA polymerase further includes an exogenousreporter moiety covalently joined thereto. For example, the exogenousreporter moiety can be a fluorescent reporter moiety. Preferably, thefluorescent reporter moiety does not substantially change excitation oremission properties following contact with any nucleotide. According toanother generally preferred embodiment, the mutant DNA polymerase can bebound to a primed template nucleic acid molecule in combination with anucleotide that is the next correct nucleotide for the primed templatenucleic acid molecule. According to another generally preferredembodiment, the mutant DNA polymerase can bind to a blocked primedtemplate nucleic acid molecule in combination with a nucleotide that isthe next correct nucleotide for the blocked primed template nucleic acidmolecule. When this is the case, the blocked primed template nucleicacid molecule can include a reversible terminator moiety on the 3′terminal nucleotide of the primer strand.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Cys (C) at position 259, and Cys (C) at position 434.According to one generally preferred embodiment, the mutant DNApolymerase can further include an N-terminal polypeptide sequenceappended to the variant of SEQ ID NO:2. Preferably, the variant sequenceis a variant of SEQ ID NO:1. According to another generally preferredembodiment, the mutant DNA polymerase further includes an exogenousreporter moiety covalently joined thereto. For example, the exogenousreporter moiety can be a fluorescent reporter moiety. When this is thecase, the fluorescent reporter moiety does not substantially changeexcitation or emission properties following contact with any nucleotide.According to another generally preferred embodiment, the mutant DNApolymerase is bound to a primed template nucleic acid molecule incombination with a nucleotide that is the next correct nucleotide forthe primed template nucleic acid molecule. According to anothergenerally preferred embodiment, the mutant DNA polymerase binds to ablocked primed template nucleic acid molecule in combination with anucleotide that is the next correct nucleotide for the blocked primedtemplate nucleic acid molecule. Preferably, the blocked primed templatenucleic acid molecule includes a reversible terminator moiety on the 3′terminal nucleotide of the primer strand.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Glu (E) at position 290, Cys (C) at position 259, andCys (C) at position 434. According to one generally preferredembodiment, the mutant DNA polymerase further includes an N-terminalpolypeptide sequence appended to the variant of the sequence of SEQ IDNO:2. Preferably, the variant sequence is a variant of SEQ ID NO:1.According to another generally preferred embodiment, the mutant DNApolymerase further includes an exogenous reporter moiety covalentlyjoined thereto. Preferably, the exogenous reporter moiety is afluorescent reporter moiety. More preferably, the fluorescent reportermoiety does not substantially change excitation or emission propertiesfollowing contact with any nucleotide. According to another generallypreferred embodiment, the mutant DNA polymerase is bound to a primedtemplate nucleic acid molecule in combination with a nucleotide that isthe next correct nucleotide for the primed template nucleic acidmolecule. According to another generally preferred embodiment, themutant DNA polymerase binds to a blocked primed template nucleic acidmolecule in combination with a nucleotide that is the next correctnucleotide for the blocked primed template nucleic acid molecule.Preferably, the blocked primed template nucleic acid molecule includes areversible terminator moiety on the 3′ terminal nucleotide of the primerstrand.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Glu (E) at position 364, and further includes Glu (E)at position 290. According to one generally preferred embodiment, themutant DNA polymerase further includes an N-terminal polypeptidesequence appended to the variant of the sequence of SEQ ID NO:2.Preferably, the variant sequence is a variant of SEQ ID NO:1. Morepreferably, the mutant DNA polymerase further includes an exogenousreporter moiety covalently joined thereto. For example, the exogenousreporter moiety can be a fluorescent reporter moiety. More preferably,the fluorescent reporter moiety does not substantially change excitationor emission properties following contact with any nucleotide. Accordingto another generally preferred embodiment, the mutant DNA polymerase isbound to a primed template nucleic acid molecule in combination with anucleotide that is the next correct nucleotide for the primed templatenucleic acid molecule. According to another generally preferredembodiment, the mutant DNA polymerase binds to a blocked primed templatenucleic acid molecule in combination with a nucleotide that is the nextcorrect nucleotide for the blocked primed template nucleic acidmolecule. Preferably, the blocked primed template nucleic acid moleculeincludes a reversible terminator moiety on the 3′ terminal nucleotide ofthe primer strand.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Glu (E) at position 364, and further includes Cys (C)at position 259 and Cys (C) at position 434. According to one generallypreferred embodiment, the mutant DNA polymerase further includes anN-terminal polypeptide sequence appended to the variant of the sequenceof SEQ ID NO:2. Preferably, the variant sequence is a variant of SEQ IDNO:1. More preferably, the mutant DNA polymerase further includes anexogenous reporter moiety covalently joined thereto. For example, theexogenous reporter moiety can be a fluorescent reporter moiety.Preferably, the fluorescent reporter moiety does not substantiallychange excitation or emission properties following contact with anynucleotide. According to another generally preferred embodiment, themutant DNA polymerase is bound to a primed template nucleic acidmolecule in combination with a nucleotide that is the next correctnucleotide for the primed template nucleic acid molecule. According toanother generally preferred embodiment, the mutant DNA polymerase bindsto a blocked primed template nucleic acid molecule in combination with anucleotide that is the next correct nucleotide for the blocked primedtemplate nucleic acid molecule. Preferably, the blocked primed templatenucleic acid molecule includes a reversible terminator moiety on the 3′terminal nucleotide of the primer strand.

In another aspect, the disclosure relates to an isolated mutant DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, wherethe variant is at least 80% identical to SEQ ID NO:2 and where thevariant includes Glu (E) at position 364, and further includes Glu (E)at position 290, Cys (C) at position 259, and Cys (C) at position 434.According to one generally preferred embodiment, the mutant DNApolymerase further includes an N-terminal polypeptide sequence appendedto the variant of the sequence of SEQ ID NO:2. Preferably, the variantsequence is a variant of SEQ ID NO:1. More preferably, the mutant DNApolymerase further includes an exogenous reporter moiety covalentlyjoined thereto. For example, the exogenous reporter moiety can be afluorescent reporter moiety. More preferably, the fluorescent reportermoiety does not substantially change excitation or emission propertiesfollowing contact with any nucleotide. According to another generallypreferred embodiment, the mutant DNA polymerase is bound to a primedtemplate nucleic acid molecule in combination with a nucleotide that isthe next correct nucleotide for the primed template nucleic acidmolecule. According to another generally preferred embodiment, themutant DNA polymerase binds to a blocked primed template nucleic acidmolecule in combination with a nucleotide that is the next correctnucleotide for the blocked primed template nucleic acid molecule.Preferably, the blocked primed template nucleic acid molecule includes areversible terminator moiety on the 3′ terminal nucleotide of the primerstrand.

In another aspect, the disclosure relates to a reaction mixture. Thereaction mixture includes a DNA polymerase that can be any of: (i) anengineered DNA polymerase that includes a variant of the sequence of SEQID NO:3, the variant being at least 80% identical to SEQ ID NO:3 andincluding an amino acid substitution mutation at one or more ofpositions K250, Q281, D355, Q425, and D532; (ii) an engineered DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, thevariant being at least 80% identical to SEQ ID NO:2 and wherein thevariant includes Glu (E) at position 290; (iii) an engineered DNApolymerase that includes a variant of the sequence of SEQ ID NO:2, thevariant being at least 80% identical to SEQ ID NO:2 and wherein thevariant includes Cys (C) at position 259, and Cys (C) at position 434;and (iv) an engineered DNA polymerase that includes a variant of thesequence of SEQ ID NO:2, the variant being at least 80% identical to SEQID NO:2 and wherein the variant includes Glu (E) at position 290, Cys(C) at position 259, and Cys (C) at position 434. Further included inthe reaction mixture are a primed template nucleic acid molecule,optionally including a reversible terminator nucleotide at a 3′-endthereof; and at least one nucleotide. According to one generallypreferred embodiment, the primed template nucleic acid molecule does notinclude the optional reversible terminator nucleotide, and the reactionmixture further includes a cation that stabilizes a ternary complex. Theternary complex includes (a) the primed template nucleic acid molecule,(b) the DNA polymerase, and (c) one of the at least one nucleotide thatis the next correct nucleotide for the primed template nucleic acidmolecule. Preferably, the cation that stabilizes ternary complexes isany of a divalent metal cation, and a trivalent metal cation. Accordingto another generally preferred embodiment, the DNA polymerase includesan exogenous detectable label. Preferably, the exogenous detectablelabel is a fluorescent label that does not substantially change itsexcitation or emission properties after binding any nucleotide.According to another generally preferred embodiment, one or more of theat least one nucleotide includes an exogenous label.

In another aspect, the disclosure relates to a kit for identifying thecognate nucleotide for a primed template nucleic acid molecule. The kitincludes a DNA polymerase that can be any of: (i) an engineered DNApolymerase that includes a variant of the sequence of SEQ ID NO:3, thevariant being at least 80% identical to SEQ ID NO:3 and including anamino acid substitution mutation at one or more of positions K250, Q281,D355, Q425, and D532; (ii) an engineered DNA polymerase that includes avariant of the sequence of SEQ ID NO:2, the variant being at least 80%identical to SEQ ID NO:2 and wherein the variant includes Glu (E) atposition 290; (iii) an engineered DNA polymerase that includes a variantof the sequence of SEQ ID NO:2, the variant being at least 80% identicalto SEQ ID NO:2 and wherein the variant includes Cys (C) at position 259,and Cys (C) at position 434; and (iv) an engineered DNA polymerase thatincludes a variant of the sequence of SEQ ID NO:2, the variant being atleast 80% identical to SEQ ID NO:2 and wherein the variant includes Glu(E) at position 290, Cys (C) at position 259, and Cys (C) at position434. The kit further includes a plurality of nucleotides or analogsthereof, and a plurality of reversible terminator nucleotides. Accordingto one generally preferred embodiment, the primed template nucleic acidincludes a blocked primer. According to another generally preferredembodiment, the primed template nucleic acid includes an extendableprimer. According to another generally preferred embodiment, the DNApolymerase includes a reporter moiety attached thereto. According toanother generally preferred embodiment, the plurality of nucleotides oranalogs thereof includes a plurality of dNTPs or analogs thereof.Preferably, the plurality of reversible terminator nucleotides includesa plurality of non-fluorescent reversible terminator nucleotides. Morepreferably, the plurality of non-fluorescent reversible terminatornucleotides is a plurality of unlabeled reversible terminatornucleotides. According to another generally preferred embodiment, thekit further includes a second polymerase that incorporates the pluralityof reversible terminator nucleotides into the primed template nucleicacid molecule. According to another generally preferred embodiment, oneor more of the plurality of nucleotides or analogs thereof includes anexogenous label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are interferometry traces for single-nucleotideexaminations, and incorporation steps; comparing results obtained usingthe CBT and TQE polymerases under different salt conditions. FIG. 1Ashows results from procedures carried out in the presence of 50 mM KCland 320 mM potassium glutamate. FIG. 1B shows results from procedurescarried out in the presence of 100 mM KCl and 320 mM potassiumglutamate. FIG. 1C shows results from procedures carried out in thepresence of 150 mM KCl and 320 mM potassium glutamate.

FIGS. 2A-2C are interferometry traces for single-nucleotideexaminations, and incorporation steps; comparing results obtained usingthe CBU and UQE polymerases under different salt conditions. FIG. 2Ashows results from procedures carried out in the presence of 50 mM KCland 320 mM potassium glutamate. FIG. 2B shows results from procedurescarried out in the presence of 100 mM KCl and 320 mM potassiumglutamate. FIG. 2C shows results from procedures carried out in thepresence of 150 mM KCl and 320 mM potassium glutamate.

FIGS. 3A-3C are interferometry traces for single-nucleotideexaminations, and incorporation steps; comparing results obtained usingthe CBT and DSA polymerases under different salt conditions. FIG. 3Ashows results from procedures carried out using an examination bufferthat included 100 mM KCl. FIG. 3B shows results from procedures carriedout using an examination buffer that included 200 mM KCl. FIG. 3C showsresults from procedures carried out using an examination buffer thatincluded 400 mM KCl.

FIGS. 4A-4C, respectively presenting results obtained using detectablylabeled CBT, TQE, and DSA polymerases, show fluorescent traces forpolymerase ternary complex formation as a function of cycling progress.Correct bases are indicated in the panels of the figures above thedifferent sets of four fluorescent traces, where each set of four peaksrepresented one complete cycle of testing four nucleotides. Between eachset of four peaks there were steps to: (a) remove reversible terminatormoieties that blocked nucleotide addition; and (b) incorporate a newreversible terminator nucleotide, thereby advancing the primer by oneposition.

FIG. 5 is a set of interferometry traces for a series ofsingle-nucleotide examination steps using the TEE polymerase and the DSApolymerase, where two rounds of examination for each polymerase areseparated by an incorporation step. Identity of the nucleotideundergoing examination is indicated below the trace (i.e., “A”represents dATP, “T” represents dTTP, “G” represents dCTP, and “C”represents dCTP). Immediately preceding each nucleotide examination stepis a step for polymerase binding in the absence of nucleotide (i.e., topermit binary complex formation). Immediately following each nucleotideexamination are steps for stripping complexes from the primed templatenucleic acid, and then regenerating the sensor tip by washing to removetraces of EDTA. Height and trajectory of the binding signals indicatethe magnitude of complex formation.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. For clarity, the following specific terms have the specifiedmeanings. Other terms are defined in other sections herein.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedin the description and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the compositions, apparatus, or methods of thepresent disclosure. At the very least, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

As used herein, “Sequencing By Binding™” refers to a sequencingtechnique wherein specific binding of a polymerase to a primed templatenucleic acid is used for identifying the next correct nucleotide to beincorporated into the primer strand of the primed template nucleic acid.The specific binding interaction precedes chemical incorporation of thenucleotide into the primer strand, and so identification of the nextcorrect nucleotide can take place either without or before incorporationof the next correct nucleotide.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”or grammatical equivalents used herein means at least two nucleotidescovalently linked together. Thus, a “nucleic acid” is a polynucleotide,such as DNA, RNA, or any combination thereof, that can be acted upon bya polymerizing enzyme during nucleic acid synthesis. The term “nucleicacid” includes single-, double-, or multiple-stranded DNA, RNA andanalogs (derivatives) thereof. Double-stranded nucleic acidsadvantageously can minimize secondary structures that may hinder nucleicacid synthesis. A double stranded nucleic acid may possess a nick or asingle-stranded gap.

As used herein, the “next correct nucleotide” (sometimes referred to asthe “cognate” nucleotide) is the nucleotide having a base complementaryto the base of the next template nucleotide. The next correct nucleotidewill hybridize at the 3′-end of a primer to complement the next templatenucleotide. The next correct nucleotide can be, but need not necessarilybe, capable of being incorporated at the 3′ end of the primer. Forexample, the next correct nucleotide can be a member of a ternarycomplex that will complete an incorporation reaction or, alternatively,the next correct nucleotide can be a member of a stabilized ternarycomplex that does not catalyze an incorporation reaction. The nextcorrect nucleotide can be a nucleotide analog. A nucleotide having abase that is not complementary to the next template base is referred toas an “incorrect” (or “non-cognate”) nucleotide. The next correctnucleotide, when participating in a ternary complex, is non-covalentlybound to the primed template nucleic acid of the ternary complex.

As used herein, the “next template nucleotide” (or the “next templatebase”) refers to the next nucleotide (or base) in a template nucleicacid that pairs with a position that is located immediately downstreamof the 3′-end of a hybridized primer. In other words, the next templatenucleotide is located immediately 5′ of the base in the template that ishybridized to the 3′ end of the primer.

As used herein, a “template nucleic acid” is a nucleic acid to be actedupon (e.g., amplified, detected or sequenced) using a method orcomposition disclosed herein.

As used herein, a “primed template nucleic acid” (or alternatively,“primed template nucleic acid molecule”) is a template nucleic acidprimed with (i.e., hybridized to) a primer, wherein the primer is anoligonucleotide having a 3′-end with a sequence complementary to aportion of the template nucleic acid. The primer can optionally have afree 5′-end (e.g., the primer being noncovalently associated with thetemplate) or the primer can be continuous with the template (e.g., via ahairpin structure). The primed template nucleic acid includes thecomplementary primer and the template nucleic acid to which it is bound.Unless explicitly stated, the primer of the primed template nucleic acidcan have either a 3′-end that is extendible by a polymerase, or a 3′-endthat is blocked from extension.

As used herein, a “blocked primed template nucleic acid” (oralternatively, “blocked primed template nucleic acid molecule”) is aprimed template nucleic acid modified to preclude or preventphosphodiester bond formation at the 3′-end of the primer. Blocking maybe accomplished, for example, by chemical modification with a blockinggroup at either the 3′ or 2′ position of the five-carbon sugar at the 3′terminus of the primer. Alternatively, or in addition, chemicalmodifications that preclude or prevent phosphodiester bond formation mayalso be made to the nitrogenous base of a nucleotide. Reversibleterminator nucleotide analogs including each of these types of blockinggroups will be familiar to those having an ordinary level of skill inthe art. Incorporation of these analogs at the 3′ terminus of a primerof a primed template nucleic acid molecule results in a blocked primedtemplate nucleic acid molecule. The blocked primed template nucleic acidincludes the complementary primer, blocked from extension at its 3′-end,and the template nucleic acid to which it is bound.

As used herein, a “nucleotide” is a molecule that includes a nitrogenousbase, a five-carbon sugar (ribose or deoxyribose), and at least onephosphate group. The term embraces, but is not limited to,ribonucleotides, deoxyribonucleotides, nucleotides modified to includeexogenous labels or reversible terminators, and nucleotide analogs.

As used herein, a “native” nucleotide refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog. Examples of native nucleotides useful for carryingout the Sequencing By Binding™ procedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate).

As used herein, a “nucleotide analog” has one or more modifications,such as chemical moieties, which replace, remove and/or modify any ofthe components (e.g., nitrogenous base, five-carbon sugar, or phosphategroup(s)) of a native nucleotide. Nucleotide analogs may be eitherincorporable or non-incorporable by a polymerase in a nucleic acidpolymerization reaction. Optionally, the 3′-OH group of a nucleotideanalog is modified with a moiety. The moiety may be a reversible orirreversible terminator of polymerase extension. The base of anucleotide may be any of adenine, cytosine, guanine, thymine, or uracil,or analogs thereof. Optionally, a nucleotide has an inosine, xanthine,hypoxanthine, isocytosine, isoguanine, nitropyrrole (including3-nitropyrrole) or nitroindole (including 5-nitroindole) base.Nucleotides may include, but are not limited to, ATP, UTP, CTP, GTP,ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dUTP, dCTP, dGTP,dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Nucleotides may alsocontain terminating inhibitors of DNA polymerase, dideoxynucleotides or2′,3′ dideoxynucleotides, which are abbreviated as ddNTPs (ddGTP, ddATP,ddTTP, ddUTP and ddCTP).

As used herein, a “blocking moiety,” when used with reference to anucleotide analog, is a part of the nucleotide that inhibits or preventsthe nucleotide from forming a covalent linkage to a second nucleotide(e.g., via the 3′-OH of a primer nucleotide) during the incorporationstep of a nucleic acid polymerization reaction. The blocking moiety of a“reversible terminator” nucleotide can be modified or removed from thenucleotide analog to allow for nucleotide incorporation. Such a blockingmoiety is referred to herein as a “reversible terminator moiety.”Exemplary reversible terminator moieties are set forth in U.S. Pat. Nos.7,427,673; 7,414,116; and 7,057,026 and PCT publications WO 91/06678 andWO 07/123744, each of which is incorporated by reference.

As used herein, a “test nucleotide” is a nucleotide being investigatedfor its ability to participate in formation of a ternary complex thatfurther includes a primed template nucleic acid and a polymerase.

As used herein, “polymerase” is a generic term for a nucleic acidsynthesizing enzyme, including but not limited to, DNA polymerase, RNApolymerase, reverse transcriptase, primase and transferase. Typically,the polymerase includes one or more active sites at which nucleotidebinding and/or catalysis of nucleotide polymerization may occur. Thepolymerase may catalyze the polymerization of nucleotides to the 3′-endof a primer bound to its complementary nucleic acid strand. For example,a polymerase can catalyze the addition of a next correct nucleotide tothe 3′ oxygen of the primer via a phosphodiester bond, therebychemically incorporating the nucleotide into the primer. Optionally, thepolymerase used in the provided methods is a processive polymerase.Optionally, the polymerase used in the provided methods is adistributive polymerase. Optionally, a polymerase need not be capable ofnucleotide incorporation under one or more conditions used in a methodset forth herein. For example, a mutant polymerase may be capable offorming a ternary complex but incapable of catalyzing nucleotideincorporation.

As used herein, a “variant” of a polypeptide reference sequence is aform or version of the polypeptide sequence that differs in somerespect. Variants can differ in amino acid sequence and can include, forexample, amino acid substitutions, additions (e.g., insertions, andextensions of termini), and deletions. A variant of a polypeptidereference sequence can include amino acid substitutions and/or internaladditions and/or deletions and/or additional amino acids at one or bothtermini of the reference sequence.

As used herein, a “polyhistidine-tag motif” is an amino acid motif inproteins that consists of six or more contiguous histidine residues, andthat facilitates binding of the proteins to an affinity support (e.g.,bead or resin) containing bound divalent nickel ions.

As used herein, a “salt providing monovalent cation” is an ioniccompound that dissociates in aqueous solution to produce cations havinga single positive charge. For example, the cations can be metal cationswhere the oxidation state is +1.

As used herein, “a glutamate salt” is an ionic compound that dissociatesin aqueous solution to produce glutamate anions.

As used herein, “biphasic” refers to a two-stage process wherein aprimed template nucleic acid is contacted with a polymerase and a testnucleotide. The first phase of the process involves contacting theprimed template nucleic acid with a polymerase in the presence of asub-saturating level of nucleotide(s), or even in the absence ofnucleotides. The term “sub-saturating,” when used in reference to ligandthat binds to a receptor (e.g., a nucleotide that binds to apolymerase), refers to a concentration of the ligand that is below thatrequired to result in at least 90% of the receptors being bound to theligand at equilibrium. For example, a sub-saturating amount ofnucleotide can yield at least 90%, 95%, 99% or more polymerases beingbound to the nucleotide. The second phase of the process involvescontacting the primed template nucleic acid from the first phase with apolymerase in the presence of a higher concentration of nucleotide(s)than used in the first phase, where the higher concentration issufficient to yield maximal ternary complex formation when a nucleotidein the reaction is the next correct nucleotide.

As used herein, “providing” a template, a primer, a primed templatenucleic acid, or a blocked primed template nucleic acid refers to thedelivery of one or many nucleic acid polymers, for example to a reactionmixture or reaction chamber. Optionally, providing a material caninclude preparation of the material in addition to its delivery.

As used herein, “monitoring” (or sometimes “measuring”) refers to aprocess of detecting a measurable interaction or binding between twomolecular species. For example, monitoring may involve detectingmeasurable interactions between a polymerase and primed template nucleicacid, typically at various points throughout a procedure. Monitoring canbe intermittent (e.g., periodic) or continuous (e.g., withoutinterruption), and can involve acquisition of quantitative results.Monitoring can be carried out by detecting multiple signals over aperiod of time during a binding event or, alternatively, by detectingsignal(s) at a single time point during or after a binding event.

As used herein, “contacting” refers to the mixing together of reagents(e.g., mixing an immobilized template nucleic acid and either a bufferedsolution that includes a polymerase, or the combination of a polymeraseand a test nucleotide) so that a physical binding reaction or a chemicalreaction may take place.

As used herein, “incorporating” or “chemically incorporating,” when usedin reference to a primed template and nucleotide, refers to the processof joining a cognate nucleotide to a primer by formation of aphosphodiester bond.

As used herein, “extension” refers to the process after anoligonucleotide primer and a template nucleic acid have annealed to oneanother, wherein a polymerase enzyme catalyzes addition of one or morenucleotides at the 3′-end of the primer. A nucleotide that is added to anucleic acid by extension is said to be “incorporated” into the nucleicacid. Accordingly, the term “incorporating” can be used to refer to theprocess of joining a nucleotide to the 3′-end of a primer by formationof a phosphodiester bond.

As used herein, a “binary complex” is an intermolecular associationbetween a polymerase and a primed template nucleic acid (e.g., blockedprimed template nucleic acid), where the complex does not include anucleotide molecule such as the next correct nucleotide.

As used herein, a “ternary complex” is an intermolecular associationbetween a polymerase, a primed template nucleic acid (e.g., blockedprimed template nucleic acid), and the next correct nucleotide moleculepositioned immediately downstream of the primer and complementary to thetemplate strand of the primed template nucleic acid or the blockedprimed template nucleic acid. The primed template nucleic acid caninclude, for example, a primer with a free 3′-OH or a blocked primer(e.g., a primer with a chemical modification on the base or the sugarmoiety of the 3′ terminal nucleotide, where the modification precludesenzymatic phosphodiester bond formation). The term “stabilized ternarycomplex” means a ternary complex having promoted or prolonged existenceor a ternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents covalent incorporation ofthe nucleotide component of the ternary complex into the primed nucleicacid component of the ternary complex.

As used herein, a “catalytic metal ion” refers to a metal ion thatfacilitates phosphodiester bond formation between the 3′-OH of a nucleicacid (e.g., a primer) and the phosphate of an incoming nucleotide by apolymerase. A “divalent catalytic metal cation” is a catalytic metal ionhaving a valence of two. Catalytic metal ions can be present atsufficiently low concentrations to stabilize formation of a complexbetween a polymerase, a nucleotide, and a primed template nucleic acid,referred to as non-catalytic concentrations of a metal ion. Catalyticconcentrations of a metal ion refer to the amount of a metal ionsufficient for polymerases to catalyze the reaction between the 3′-OHgroup of a nucleic acid (e.g., a primer) and the phosphate group of anincoming nucleotide.

As used herein, a “non-catalytic metal ion” refers to a metal ion that,when in the presence of a polymerase enzyme, does not facilitatephosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. Typically, the non-catalytic metal ion is acation. A non-catalytic metal ion may inhibit phosphodiester bondformation by a polymerase, and so may stabilize a ternary complex bypreventing nucleotide incorporation. Non-catalytic metal ions mayinteract with polymerases, for example, via competitive binding comparedto catalytic metal ions. A “divalent non-catalytic metal ion” is anon-catalytic metal ion having a valence of two. Examples of divalentnon-catalytic metal ions include, but are not limited to, Ca²⁺, Zn²⁺,Ni²⁺, and Sr²⁺. The trivalent Eu³⁺ and Tb³⁺ ions are non-catalytic metalions having a valence of three.

As used herein an “exogenous label” refers to a detectable chemicalmoiety that has been added to another entity, such as a nucleotide,polymerase (e.g., a DNA polymerase) or other sequencing reagent setforth herein. While a native dNTP may have a characteristic limitedfluorescence profile, the native dNTP does not include any addedcolorimetric or fluorescent moiety. Conversely, a dATP(2′-deoxyadenosine-5′-triphosphate) molecule modified to include achemical linker and fluorescent moiety attached to the gamma phosphatewould be said to include an exogenous label because the attachedchemical components are not ordinarily a part of the nucleotide. Ofcourse, chemical modifications to add detectable labels to nucleotidebases also would be considered exogenous labels. Likewise, a DNApolymerase modified to include a conformationally sensitive fluorescentdye that changes its properties upon nucleotide binding also would besaid to include an exogenous label because the label is not ordinarily apart of the polymerase.

As used herein, “unlabeled” refers to a molecular species free of addedor exogenous label(s) or tag(s). Of course, unlabeled nucleotides willnot include either of an exogenous fluorescent label, or an exogenousRaman scattering tag. A native nucleotide is another example of anunlabeled molecular species. An unlabeled molecular species can excludeone or more of the labels set forth herein or otherwise known in the artrelevant to nucleic acid sequencing or analytical biochemistry.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g., due to porosity) but will typically be sufficiently rigid thatthe substrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, a “flow cell” is a reaction chamber that includes one ormore channels that direct fluid in a predetermined manner to conduct adesired reaction. The flow cell can be coupled to a detector such that areaction occurring in the reaction chamber can be observed. For example,a flow cell can contain primed template nucleic acid molecules, forexample, tethered to a solid support, to which nucleotides and ancillaryreagents are iteratively applied and washed away. The flow cell caninclude a transparent material that permits the sample to be imagedafter a desired reaction occurs. For example, a flow cell can include aglass or plastic slide containing small fluidic channels through whichpolymerases, dNTPs and buffers can be pumped. The glass or plasticinside the channels can be decorated with one or more primed templatenucleic acid molecules to be sequenced. An external imaging system canbe positioned to detect the molecules on the surface of the glass orplastic. Reagent exchange in a flow cell is accomplished by pumping,drawing, or otherwise “flowing” different liquid reagents through theflow cell. Exemplary flow cells, methods for their manufacture andmethods for their use are described in US Pat. App. Publ. Nos.2010/0111768 A1 or 2012-0270305 A1; or WO 05/065814, each of which isincorporated by reference herein.

As used herein, a “reaction vessel” is a container that isolates onereaction (e.g., a binding reaction; an incorporation reaction; etc.)from another, or that provides a space in which a reaction can takeplace. Non-limiting examples of reaction vessels useful in connectionwith the disclosed technique include: flow cells, wells of a multiwellplate; microscope slides; tubes (e.g., capillary tubes); etc. Featuresto be monitored during binding and/or incorporation reactions can becontained within the reaction vessel.

As used herein, a “kit” is a packaged unit containing one or morecomponents that can be used for performing detection and/or sequencingreactions using an engineered DNA polymerase, as disclosed herein.Typical kits may include packaged combinations, in one or morecontainers or vials, of reagents to be used in the procedure.

DETAILED DESCRIPTION Introduction and Overview

The Sequencing By Binding™ method disclosed by Vijayan et al., inpublished U.S. patent application publication number 2017/0022553A1benefits from reduced polymerase binding to primed template nucleic acidin the absence of cognate nucleotide (e.g., whether in the absence ofany nucleotide, or in the presence of only non-cognate nucleotide).Different approaches have proven useful for reducing the magnitude ofthis binary complex formation, while at the same time stabilizingternary complexes that include primed template nucleic acid, polymerase,and the cognate nucleotide. For example, some approaches rely onmanipulation of salt concentrations or the manner of deliveringpolymerase to the primed template to enhance this discrimination.

Polymerases that exhibit reduced nucleotide-independent interaction withDNA templates would be useful tools in Sequencing By Binding™procedures. This is particularly true when labeled polymeraseinteraction with a primed template nucleic acid is monitored as asurrogate for cognate nucleotide identification. Binary complexformation confounds identification of cognate nucleotide when signal dueto cognate nucleotide identification is not substantially greater thansignal due to polymerase binding in the presence of non-cognatenucleotide (i.e., conditions of weak discrimination). Below there aredescribed engineered DNA polymerases that are useful for enhancingdetection of cognate nucleotides by reducing signals associated withpolymerase binding in the absence of cognate nucleotide. The engineeredpolymerases can have other uses as will be recognized by those skilledin the art in view of the teaching set forth herein.

DESCRIPTION OF VARIOUS EMBODIMENTS

Described below are the preparation of DNA polymerase I (pol I) largefragment mutants from a thermostable family strain of Bacillusstearothermophilus (Bst-f), and from Bacillus subtilis (Bsu-f), wherethe mutants form ternary complexes with cognate nucleotides whileexhibiting reduced DNA-binding affinity in dynamic equilibrium bindingassays. Both of the Bst-f and Bsu-f enzymes are family A polymeraseshaving homology to other well-characterized, high fidelity polymerases,including E. coli DNA pol I (KF), and T. aquaticus DNA pol I (Taq).These polymerases share certain conserved protein sequence motifs, butare distinguished by certain non-conserved regions.

The parent enzyme (“CBT”) used for preparing certain reduced DNAaffinity polymerases was an engineered version of the Bst polymerase.The polypeptide sequence of the CBT enzyme had been modified withrespect to cysteine content, and by addition of N-terminal sequencesthat facilitated protein purification and processing. More specifically,the polypeptide sequence identified as SEQ ID NO:1 included a modifiedN-terminus having: (1) an engineered polyhistidine-tag motif atpositions 5-10; (2) a thrombin cleavage site between positions Arg17 andGly18; and (3) a cysteine residue at position 23. The naturallyoccurring Bst polymerase sequence extended from position 27 to theC-terminus (subject to replacement of naturally occurring cysteineresidues). It is to be understood that engineered polymerases inaccordance with the disclosure optionally include or omit the N-terminalmodifications that do not substantially affect DNA affinity of thepolymerase. For example, useful polymerases can be constructed on aparent scaffold of SEQ ID NO:2 (i.e., the polypeptide sequence of SEQ IDNO:1 following thrombin cleavage) or SEQ ID NO:3 (i.e., the proteinexpression product of the cysteine-substituted Bst-f polymerase).Examples of variant polypeptide sequences relative to each of thesescaffolds are presented in Table 1. Nucleic acid modifications used toencode the reduced DNA affinity polymerases were prepared usingsite-directed mutagenesis and prokaryotic expression cloning vectorsthat will be familiar to those having an ordinary level of skill in theart.

The Bacillus DNA polymerase large fragment (i.e., the C-terminalfragment commonly used in crystal structure analysis; and lacking 5′-3′exonuclease activity) of SEQ ID NO:4 served as the scaffold forconstruction of the engineered polymerases derived from the CBTconstructs, as disclosed herein. The engineered CBT polymerase of SEQ IDNO:3 differs from the sequence of the wild type Bacillus polymeraselarge fragment of SEQ ID NO:4 by substitution of two Cys residues (i.e.,at positions 90 and 547) by Ala residues. The sequence of theCys-substituted polymerase of SEQ ID NO:2 differs from the engineeredpolymerase of SEQ ID NO:3 by further including an amino-terminalsequence of amino acids given by SEQ ID NO:5. Likewise, the sequence ofthe N-terminal modified and Cys-substituted polymerase of SEQ ID NO:1differs from the engineered polymerase of SEQ ID NO:3 by furtherincluding an amino-terminal sequence of amino acids given by SEQ IDNO:6. Since the N-terminal modifications employed in preparation of theengineered DNA polymerases described herein (i.e., SEQ ID NO:5 and SEQID NO:6) are not known to affect enzymatic activities, useful engineeredDNA polymerases can be described in the context of the base scaffold ofSEQ ID NO:3.

The parent enzyme (“CBU”) used for preparing anotherspecificity-enhanced polymerase was an engineered version of the Bsupolymerase. The polypeptide sequence of the CBU enzyme had been modifiedwith respect to cysteine content, and N-terminal sequences thatfacilitated protein purification and processing. More specifically, thepolypeptide sequence identified as SEQ ID NO:13 included a modifiedN-terminus having: (1) an engineered polyhistidine tag motif atpositions 5-10; (2) a thrombin cleavage site between positions Arg17 andGly18; and (3) a cysteine residue at position 23. The naturallyoccurring Bsu polymerase sequence extended from position 27 to theC-terminus. It is to be understood that engineered polymerases inaccordance with the disclosure optionally include or omit the N-terminalmodifications that do not substantially affect DNA affinity of thepolymerase. For example, useful polymerases can be constructed on aparent scaffold of SEQ ID NO:12 (i.e., essentially the polypeptidesequence of SEQ ID NO:13 following thrombin cleavage). Variantpolypeptide sequences corresponding to useful specificity-enhancedpolymerases (e.g., “UQE” mutant polymerases) relative to each of thesescaffolds are presented in Table 1. Nucleic acid modifications used toencode the UQE polymerase were prepared using site-directed mutagenesisand prokaryotic expression cloning vectors that will be familiar tothose having an ordinary level of skill in the art.

Sequence Comparison, Identity, and Homology

The term “identical,” in the context of two or more nucleic acid orpolypeptide sequences, refers to two or more sequences or subsequencesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (or other algorithms available to persons of skill) or by visualinspection. The term “percent identity,” in the context of two or morenucleic acid or polypeptide sequences, refers to two or more sequencesor subsequences that have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumcorrespondence, as measured using one of the sequence comparisonalgorithms described below (or other algorithms available to persons ofskill) or by visual inspection. By convention, amino acid additions,substitutions, and deletions within an aligned reference sequence areall differences that reduce the percent identity in an equivalentmanner. Additional amino acids present at the N- or C-terminus of apolynucleotide compared to the reference have no effect on percentidentity scoring for aligned regions. For example, alignment of a 105amino acid long polypeptide to a reference sequence 100 amino acids longwould have a 100% identity score if the reference sequence fully wascontained within the longer polynucleotide with no amino aciddifferences. A single amino acid difference (addition, deletion orsubstitution) between the two sequences within the 100-amino acid spanof the aligned reference sequence would mean the two sequences were 99%identical.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding a polymerase, or the aminoacid sequence of a polymerase) refers to two or more sequences orsubsequences that have at least about 60%, about 80%, about 90-95%,about 98%, about 99% or more nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usinga sequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably, the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

Proteins and/or protein sequences are “homologous” when they arederived, naturally or artificially, from a common ancestral protein orprotein sequence. Similarly, nucleic acids and/or nucleic acid sequencesare homologous when they are derived, naturally or artificially, from acommon ancestral nucleic acid or nucleic acid sequence. Homology isgenerally inferred from sequence similarity between two or more nucleicacids or proteins (or sequences thereof). The precise percentage ofsimilarity between sequences that is useful in establishing homologyvaries with the nucleic acid and protein at issue, but as little as 25%sequence similarity over 50, 100, 150 or more residues is routinely usedto establish homology. Higher levels of sequence similarity, e.g., 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used toestablish homology. Methods for determining sequence similaritypercentages (e.g., BLASTP and BLASTN using default parameters) aredescribed herein and are generally available.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyCurrent Protocols in Molecular Biology, Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., supplemented through 2004).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Substitution or replacement of one amino acid for another (i.e.,so-called “substitution mutations”) can be used for modifying functionalproperties of engineered DNA polymerases. In certain embodiments, asubstitution mutation comprises a mutation to a residue having anonpolar side chain. Amino acids having nonpolar side chains are wellknown in the art and include, for example: glycine (Gly or G), alanine(Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile orI), methionine (Met or M), phenylalanine (Phe or F), tryptophan (Trp orW), and proline (Pro or P). In certain embodiments, a substitutionmutation comprises a mutation to a residue having a polar side chain.Amino acids having polar side chains are well known in the art andinclude, for example: serine (Ser or S), threonine (Thr or T), cysteine(Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine(Gln or Q). In certain embodiments, a substitution mutation comprises amutation to a residue having an acidic side chain. Amino acids havingacidic side chains are well known in the art and include, for example:aspartate (Asp or D) and glutamate (Glu or E). In certain embodiments, asubstitution mutation comprises a mutation to a residue having a basicside chain. Amino acids having basic side chains are well known in theart and include, for example: lysine (Lys or K), arginine (Arg or R),and histidine (His or H).

A summary of primary amino acid sequence features of polymerases used inthe procedures disclosed below are presented in Table 1.

TABLE 1 Summary of Key Amino Acid Substitutions Mutant Name FeaturePosition CBT Cys-substituted Bst enzyme SEQ ID NO: 1 with N-terminalmodifications SEQ ID NO: 2 Cys-substituted Bst enzyme SEQ ID NO: 3Engineered Bst (Cys removed) TDE Crippled polymerase does not D to E at381 of SEQ ID NO: 1; or catalyze Mg²⁺-dependent D to E at 364 of SEQ IDNO: 2; or incorporation D to E at 355 of SEQ ID NO: 3 BDE Crippledpolymerase does not D to E at 558 of SEQ ID NO: 1 or catalyzeMg²⁺-dependent D to E at 541 of SEQ ID NO: 2 or incorporation D to E at532 of SEQ ID NO: 3 TQE Polymerase that discriminates Q to E at 307 ofSEQ ID NO: 1 or between binary and ternary Q to E at 290 of SEQ ID NO: 2or complexes under lower salt Q to E at 281 of SEQ ID NO: 3 conditions,and exhibits reduced DNA binding absent cognate nucleotide DSAPolymerase that discriminates K to C at 276 of SEQ ID NO: 1; and betweenbinary and ternary Q to C at 451 of SEQ ID NO: 1 complexes under lowersalt K to C at 259 of SEQ ID NO: 2; and conditions, and exhibits Q to Cat 434 of SEQ ID NO: 2 reduced DNA binding absent K to C at 250 of SEQID NO: 3; and cognate nucleotide Q to C at 425 of SEQ ID NO: 3 TEECombination of TQE and Q to E at 307 of SEQ ID NO: 1; and DSAmodifications; exhibits K to C at 276 of SEQ ID NO: 1; and somewhatimproved Q to C at 451 of SEQ ID NO: 1 discrimination relative to Q to Eat 290 of SEQ ID NO: 2; and DSA K to C at 259 of SEQ ID NO: 2; and Q toC at 434 of SEQ ID NO: 2 Q to E at 281 of SEQ ID NO: 3; and K to C at250 of SEQ ID NO: 3; and Q to C at 425 of SEQ ID NO: 3 DEA Combinationof TDE and D to E at 381 of SEQ ID NO: 1 TQE modifications; crippled Qto E at 307 of SEQ ID NO: 1 DNA polymerase does not D to E at 364 of SEQID NO: 2 catalyze Mg²⁺-dependent Q to E at 290 of SEQ ID NO: 2incorporation; exhibits D to E at 355 of SEQ ID NO: 3 reduced DNAbinding absent Q to E at 281 of SEQ ID NO: 3 cognate nucleotide TSACombination of TDE and D to E at 381 of SEQ ID NO: 1 DSA modifications;crippled K to C at 276 of SEQ ID NO: 1; and DNA polymerase does not Q toC at 451 of SEQ ID NO: 1 catalyze Mg²⁺-dependent D to E at 364 of SEQ IDNO: 2 incorporation; discriminates K to C at 259 of SEQ ID NO: 2; andunder low salt conditions Q to C at 434 of SEQ ID NO: 2 D to E at 355 ofSEQ ID NO: 3 K to C at 250 of SEQ ID NO: 3; and Q to C at 425 of SEQ IDNO: 3 TRI Combination of TDE, TQE, D to E at 381 of SEQ ID NO: 1 and DSAmodifications; Q to E at 307 of SEQ ID NO: 1 crippled DNA polymerase Kto C at 276 of SEQ ID NO: 1; and does not catalyze Mg²⁺- Q to C at 451of SEQ ID NO: 1 dependent incorporation; D to E at 364 of SEQ ID NO: 2exhibits reduced DNA Q to E at 290 of SEQ ID NO: 2 binding absentcognate K to C at 259 of SEQ ID NO: 2; and nucleotide; discriminates Qto C at 434 of SEQ ID NO: 2 under low salt conditions D to E at 355 ofSEQ ID NO: 3 Q to E at 281 of SEQ ID NO: 3 K to C at 250 of SEQ ID NO:3; and Q to C at 425 of SEQ ID NO: 3 CBU Cys-substituted Bsu enzyme N/Awith N-terminal modifications UQE Q to E 288 of SEQ ID NO: 12; or 314 ofSEQ ID NO: 13

Engineered Polymerases Incorporating Combinations of Mutated Positions

Combinations of mutated positions within the disclosed scaffolds of SEQID NOS:1-3 are embraced by the present disclosure, and can, for example,be used in connection with Sequencing By Binding™ protocols. Morespecifically, the engineered polynucleotide sequence of SEQ ID NO:3optionally can further include one or more N-terminal amino acids, andthe resulting polypeptide can further include at least one changed aminoacid at a corresponding position in the sequence of SEQ ID NO:3. Forexample, polypeptides having the amino acid sequences of SEQ ID NO:2 andSEQ ID NO:1 (each of which fully contains the sequence of SEQ ID NO:3)can be used in Sequencing By Binding™ protocols, and optionally caninclude amino acid substitutions or replacements at the correspondingposition of SEQ ID NO:3. For clarity, the last position of SEQ ID NO:3(a Lys residue at position 578) corresponds to the last position of eachof SEQ ID NO:1 (position 604) and SEQ ID NO:2 (position 587). Thus, thesequences of SEQ ID Nos:1-3 all align with each other.

Several exemplary positions within the disclosed engineered polypeptidescaffolds of SEQ ID Nos:1-3 are disclosed herein. Other positionsoptionally can be changed, and still fall within the scope of thedisclosure. Preferably, at least one and up to 10 amino acids within thesequence of SEQ ID NO:3 (including the sequence found within thesequences of SEQ ID NOs:1-2) are substituted or replaced by differentamino acids. Illustrative positions within the polypeptide sequence ofSEQ ID NO:3 that can be substituted to provide desired activity includeposition numbers: 250, 281, 355, 425, and 532. In particularly preferredembodiments, all different combinations of these positions optionallycan be mutated or replaced (e.g., in combinations of 2, 3, 4, or evenall 5 substitutions). Illustrative embodiments of these combinations aredisclosed herein. Combinations of up to 10 substituted positions arepreferred and embraced by the disclosure. However, it will be understoodthat a variant of a sequence set forth herein can include more than 10substitutions, and are within the scope of the present disclosure.

All combinations of the amino acid replacements disclosed herein (e.g.,enumerated in Table 1) fall within the scope of the disclosure, andapply to the polypeptide scaffolds of each of SEQ ID Nos:1-3 (i.e.,where a replacement in the sequence of SEQ ID NO:3 translates to thescaffolds of SEQ ID NO:1-2, by the correspondence set forth in Table 1).In the context of the scaffold of SEQ ID NO:3, unique combinations oftwo amino acid replacements are found among the following permutations:D to E at 355 in combination with any of: D to E at 532, Q to E at 281,K to C at 250, or Q to C at 425; D to E at 532 in combination with anyof: Q to Eat 281, K to C at 250, or Q to C at 425; Q to C at 281 incombination with any of: K to C at 250, or Q to C at 425; or K to C at250 in combination with Q to C at 425. Unique combinations of threeamino acid replacements are found among the following permutations: thecombination of D to E at 355, and D to E at 532 in further combinationwith any of: Q to E at 281, K to C at 250, or Q to C at 425; thecombination of D to E at 532, and Q to E at 281 in further combinationwith any of: D to E at 355, K to C at 250, or Q to C at 425; thecombination of Q to Eat 281, and K to C at 250 in further combinationwith any of: D to E at 355, D to E at 532, or Q to C at 425; thecombination of K to C at 250, Q to C at 425 in further combination withany of: D to E at 355, D to E at 532, or Q to E at 281; the combinationof D to E at 355 and Q to E at 281 in further combination with any of: Dto Eat 532, K to Cat 250, or Q to C at 425; the combination of D to Eat355 and K to C at 250 in further combination with any of: D to E at 532,Q to E at 281, or Q to C at 425; the combination of D to E at 355 and Qto C at 425 in further combination with any of: D to E at 532, Q to Eat281, or K to Cat 250; the combination of D to Eat 532 and K to Cat 250in further combination with any of: D to E at 355, Q to E at 281, or Qto C at 425; the combination of D to E at 532 and Q to C at 425 infurther combination with any of: D to E at 355, Q to Eat 281, or K to Cat 250; or the combination of Q to Eat 281 and Q to Cat 425 in furthercombination with any of: D to E at 355, D to E at 532, or K to C at 250.Unique combinations of four amino acid replacements are found among thefollowing permutations: the combination of D to E at 355, D to E at 532,Q to E at 281 in further combination with any of: K to C at 250, or Q toC at 425; the combination of D to E at 532, Q to Eat 281, K to C at 250in further combination with any of: D to E at 355, or Q to C at 425; thecombination of Q to E at 281, K to C at 250, and Q to C at 425 infurther combination with any of: D to E at 355, or D to E at 532; thecombination of D to E at 355, Q to E at 281, and K to C at 250 infurther combination with any of: D to E at 532, or Q to C at 425; thecombination of D to E at 355, Q to E at 281, and Q to C at 425 infurther combination with any of: D to E at 532, or K to C at 250; thecombination of D to E at 355, D to E at 532, and K to C at 250 infurther combination with any of: Q to E at 281, or Q to C at 425; or thecombination of D to E at 355, D to E at 532, and Q to C at 425 infurther combination with any of: Q to E at 281, or K to C at 250. Thecombination of all five amino acid replacements is represented by D to Eat 355, D to E at 532, Q to E at 281, K to C at 250, and Q to C at 425.Each unique combination of amino acid replacements in the scaffold ofSEQ ID NO:3, or the scaffold of SEQ ID NO:1 or SEQ ID NO:2 which containthe sequence of SEQ ID NO:3 are embraced by the present disclosure.

Polymerases exhibiting advantageous features include: (1) thoseclassified as “crippled” DNA polymerases; and/or (2) polymerasesexhibiting reduced affinity for primed template nucleic acids in theabsence of cognate nucleotide, and an ability to discriminate betweencognate and non-cognate nucleotides under low salt conditions. Each ofthese features can sort independently (e.g., combination mutants canpossess more than one of these features). Interestingly, the independentmutations characteristic of the TQE and DSA polymerases affectedsubstantially the same activities (i.e., low salt discriminatorycapability, and reduced DNA binding) of the polymerase. Engineered DNApolymerases lacking the capacity to promote Mg²⁺-dependent incorporationof cognate nucleotides into primed template nucleic acids (i.e.,so-called “crippled” DNA polymerases) also are disclosed in commonlyassigned U.S. patent application Ser. No. 15/581,822, published as US2017/030135 A1, the disclosure of which is incorporated by referenceherein in its entirety. The present disclosure particularly embracesengineered DNA polymerases comprising amino acid replacement orsubstitution mutations of these crippled DNA polymerases in combinationwith each other, and in combination with other replacement orsubstitution mutations disclosed herein. Likewise, combinations ofdifferent substitution mutations leading to reduced affinity ofpolymerase for primed template nucleic acid in the absence of cognatenucleotide can be combined with each other, or with other replacement orsubstitution mutations, such as those described herein.

Useful Recombinant DNA and Protein Expression Techniques

Conventional recombinant DNA cloning techniques can be used to prepareconstructs for transformation or transfection (“transformation”hereafter) and expression of nucleic acids encoding engineeredpolymerases in accordance with the disclosure. Nucleic acid constructsencoding polymerase fragments were used in combination with syntheticoligonucleotides, standard PCR techniques, and vector ligation tointroduce the site-directed mutations needed to produce thepolynucleotide sequences disclosed herein. The different constructs wereligated into a plasmid expression vector, and the plasmid constructintroduced into an E. coli host using standard transformationtechniques. Preferred expression vectors include a T7 promoter sequenceupstream of the polymerase-encoding insert, where the T7 promoter isinducible by IPTG or by co-expression of a T7 RNA polymerase. Expressedproteins included a polyhistidine-tag motif that facilitated binding ofthe recombinant protein to a nickel-based resin column as part of thepurification process.

Embraced by the present description are nucleic acid molecules encodingaltered polymerase enzymes. In accordance with various embodiments, adefined nucleic acid includes not only the identical nucleic acid butalso any minor base variations including, in particular, substitutionsin cases which result in a synonymous codon (a different codonspecifying the same amino acid residue) due to the degenerate code inconservative amino acid substitutions. The term “nucleic acid sequence”can also include the complementary sequence to any single strandedsequence given regarding base variations. Nucleic acid moleculesencoding the engineered DNA polymerases described herein may also beincluded in a suitable expression vector to express the polymeraseproteins encoded therefrom in a suitable host. Such an expression vectorincludes a vector having a nucleic acid according to the embodimentspresented herein operably linked to regulatory sequences, such aspromoter regions, that are capable of effecting expression of said DNAfragments. The term “operably linked” refers to a juxtaposition whereinthe components described are in a relationship permitting them tofunction in their intended manner. Such vectors may be transformed intoa suitable host cell to provide for the expression of a recombinantprotein. Regulatory elements required for expression include promotersequences to bind RNA polymerase and to direct an appropriate level oftranscription initiation and also translation initiation sequences forribosome binding. For example, a bacterial expression vector may includea promoter such as the lac promoter and for translation initiation theShine-Dalgarno sequence and AUG start codon. Similarly, a eukaryoticexpression vector may include a heterologous or homologous promoter forRNA polymerase II, a downstream polyadenylation signal, the start codonAUG, and a termination codon for detachment of the ribosome. Suchvectors may be obtained commercially or be assembled from the sequenceswell known in the art.

Covered nucleic acid molecules may encode a mature protein or a proteinhaving a prosequence, including that encoding a leader sequence on thepreprotein which is then cleaved by the host cell to form a matureprotein. The vectors may be, for example, plasmid, virus or phagevectors provided with an origin of replication, and optionally apromoter for the expression of said nucleotide and optionally aregulator of the promoter. The vectors may contain one or moreselectable markers, such as, for example, an antibiotic resistance gene.

Recombinant polymerase proteins can be, and indeed were, furtherpurified and concentrated using conventional laboratory techniques thatwill be familiar to those having an ordinary level of skill in the art.Purified polymerase samples were stored at −80° C. until being used.

Accordingly, the present disclosure provides a nucleic acid constructencoding one or more of the protein sequences set forth herein. Inparticular embodiments, the nucleic acid construct is a plasmid orvector. The nucleic acid construct can include elements that allowreplication of the construct, biological selection for the constructand/or expression of the one or more proteins encoded by the construct.Suitable vector backbones include, for example, those routinely used inthe art such as plasmids, artificial chromosomes, BACs, or PACs.Numerous vectors and expression systems are commercially available fromsuch corporations as Novagen (Madison, Wis.), Clonetech (Pal Alto,Calif.), Stratagene (La Jolla, Calif.), and ThermoFisher (Waltham,Mass.). Vectors typically contain one or more regulatory regions.Regulatory regions include, without limitation, promoter sequences,enhancer sequences, response elements, protein recognition sites,inducible elements, protein binding sequences, 5′ and 3′ untranslatedregions (UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns.

The present disclosure also provides recombinant organisms that includea nucleic acid construct that encodes one or more of the proteinsequences set forth herein. A recombinant organism of the presentdisclosure can be configured to express one or more polymerase having asequence set forth herein. Furthermore, the present disclosure providesa recombinant organism that comprises a polymerase having a sequence setforth herein.

In another embodiment, a cultured cell is provided that is transformedor transfected (“transformed” hereafter) with a vector comprising anucleic acid construct described herein. In this regard, a cell issuccessfully transformed with a vector when the transcription machineryof the intact cell has access to the nucleic acid template for theproduction of mRNA. Protocols to facilitate transformation of vectorsinto cells are well known in the art. Also provided herein are theprogeny of a cultured cell that was stably transformed with the vectoras described above. Such progeny will contain copies of the vectorwithout having undergone the transformation protocol and are capable oftranscribing the nucleic acids contained in vector under the control ofan expression control sequence. Techniques utilizing cultured cellstransformed with expression vectors to produce quantities ofpolypeptides are well known in the art.

Useful Polymerase Labeling and Processing Techniques

Depending on the application, engineered polymerases according to thedisclosure may be either labeled with a detectable label, or unlabeled.Unlabeled polymerases may be used in label-free systems, oralternatively can be used in conjunction with detectably labelednucleotides and/or template nucleic acids. Detectably labeledpolymerases can be used in combination with unlabeled nucleotides, orunlabeled primer or template nucleic acids for cognate nucleotideidentification. Of course, the engineered polymerases may simply be usedfor synthesizing DNA strands in template-dependent DNA synthesisreactions.

Engineered polymerases can be covalently modified, post-purification, tocontain a fluorescent moiety. For example, a fluorescent moiety can bejoined to the free sulfhydryl of a Cys residue located toward theN-terminal ends of a protein. To demonstrate the technique, a Cy-5fluorescent label chemically activated as a maleimide ester was joinedto the free thiol functional group of the N-terminal region Cys residueusing standard protein labeling techniques. While use of labeledengineered polymerases was demonstrated using a fluorescent label, manyother types of labels also may be used. Moreover, other attachmentchemistries can be used as well.

Alternative labels may be used for labeling engineered polymerases inaccordance with the disclosure. Labels attached to the polymerases canbe detectable by changes in any of: refractive index, charge detection,Raman scattering detection, ellipsometry detection, pH detection, sizedetection, mass detection, surface plasmon resonance, guided moderesonance, nanopore optical interferometry, whispering gallery moderesonance, nanoparticle scattering, photonic crystal, quartz crystalmicrobalance, bio-layer interferometry, vibrational detection, pressuredetection and other label free detection schemes that detect the addedmass or refractive index due to polymerase binding in a closed-complexwith a template nucleic acid, and the like. Further examples of usefullabels are set forth in sections below.

Polymerases in accordance with the disclosure can be subjected tofurther post-purification processing to enhance functional properties ormodify structure. This can involve chemical modification and/orenzymatic processing. Optionally, a portion of the engineered polymeraseis cleaved from the remainder of the polypeptide, and removed.

During performance of a Sequencing By Binding™ procedure, the engineeredpolymerase used to identify cognate nucleotide optionally can be usedfor incorporating the same or a different type of nucleotide. Forexample, in some embodiments it is preferable to remove labeledpolymerase and nucleotide following an examination step, and then toreplace that first polymerase and nucleotide with the same or differentnucleotide and a different polymerase. Optionally, the replacednucleotide can be a reversible terminator nucleotide (e.g., an unlabeledreversible terminator nucleotide).

Allele-Specific Capture Using Engineered Polymerases: General Aspects

Engineered DNA polymerases in accordance with the disclosure can be usedto perform allele-specific capture of target nucleic acids, for exampleas described in commonly owned U.S. patent application identified bySer. No. 15/701,358 and its priority provisional application having Ser.No. 62/448,730, the entire disclosures of which are incorporated byreference herein. More particularly, engineered DNA polymerases can beused for selecting or capturing nucleic acids having target alleles ofinterest. For example, a stabilized ternary complex can be formedbetween a polymerase, target allele and cognate nucleotide for theallele. Polymerase specificity allows a target allele to be separatedfrom other nucleic acids, including for example, other alleles thatdiffer from the target allele by a single nucleotide.

In one embodiment, a method for separating a target allele from amixture of nucleic acids includes the step of (a) providing a mixture ofnucleic acids in fluidic contact with a stabilized ternary complex thatis attached to a solid support. The stabilized ternary complex includesan engineered polymerase, a primed nucleic acid template, and a nextcorrect nucleotide. The template includes a target allele, where thenext correct nucleotide is a cognate nucleotide for the target allele.The stabilized ternary complex can be attached to the solid support viaa linkage between the polymerase and the solid support, or via a linkagebetween the next correct nucleotide and the solid support. There also isthe step of (b) separating the solid support from the mixture of nucleicacids, thereby separating the target allele from the mixture of nucleicacids.

In another embodiment, a method for separating a plurality of targetalleles from a mixture of nucleic acids includes the step of (a)providing a mixture of nucleic acids in fluidic contact with a pluralityof stabilized ternary complexes that are solid support-attached. Thestabilized ternary complexes each include an engineered polymerase, aprimed nucleic acid template, and a next correct nucleotide. Thetemplate includes a target allele, and the next correct nucleotide is acognate nucleotide for the target allele. Each of the stabilized ternarycomplexes can be attached to the solid support via a linkage between thepolymerase and the solid support, or via a linkage between the nextcorrect nucleotide and the solid support. There also is the step of (b)separating the solid support from the mixture of nucleic acids, therebyseparating the target alleles from the mixture of nucleic acids.

In another embodiment, a method for separating a first allele of a locusfrom a second allele at the locus includes the step of (a) providing amixture including the second allele in fluidic contact with a stabilizedternary complex that is attached to a solid support. The stabilizedternary complex includes an engineered polymerase, a primer hybridizedto a nucleic acid template, and a next correct nucleotide. The templateincludes the first allele. The next correct nucleotide is a cognatenucleotide for the first allele, or the 3′-end of the primer includes acognate nucleotide for the first allele. The stabilized ternary complexcan be attached to the solid support via a linkage between thepolymerase and the solid support, or via a linkage between the nextcorrect nucleotide and the solid support. There also is the step of (b)separating the solid support from the mixture of nucleic acids, therebyseparating the first allele from the second allele.

In another embodiment, a method for separating first alleles at aplurality of loci from second alleles at the plurality of loci,respectively, includes the step of (a) providing a mixture of the secondalleles at the plurality of loci, respectively, in fluidic contact witha plurality of stabilized ternary complexes that are solidsupport-attached. The stabilized ternary complexes each include anengineered DNA polymerase, a primed nucleic acid template, and a nextcorrect nucleotide. The template includes a first allele, where the nextcorrect nucleotide is a cognate nucleotide for the first allele, or the3′-end of the primer includes a cognate nucleotide for the first allele.Each of the stabilized ternary complexes is attached to the solidsupport via a linkage between the polymerase and the solid support, orvia a linkage between the next correct nucleotide and the solid support.There also is the step of (b) separating the solid support from themixture of nucleic acids, thereby separating the first alleles from thesecond alleles at the plurality of loci.

Genotyping Using Engineered Polymerases: General Aspects

Engineered DNA polymerases in accordance with the disclosure can be usedto perform genotyping by polymerase binding, for example as described incommonly owned U.S. patent application identified by Ser. No. 15/701,373and its priority provisional application having Ser. No. 62/448,630, theentire disclosures of which are incorporated by reference herein. Forexample, a ternary complex can be formed between an engineered DNApolymerase, a primed template encoding a target single nucleotidepolymorphism (SNP) allele and a cognate nucleotide for the SNP allele.Detection of the ternary complex will result in selective detection ofthe SNP allele, compared to a non-target SNP allele at the same locus,because the cognate nucleotide is selective for the target SNP whenforming a ternary complex with the polymerase.

In one embodiment, a method for identifying target alleles in a mixtureof nucleic acids includes the step of (a) providing an array offeatures, where different locus-specific primers are attached atdifferent features of the array. There also is the step of (b)contacting the array with a plurality of nucleic acid templates,engineered DNA polymerases and nucleotides to form a plurality ofstabilized ternary complexes at a plurality of the features. Thestabilized ternary complexes each include an engineered DNA polymerase,a template nucleic acid including a target allele of a locus, alocus-specific primer of the array hybridized to the locus, and a nextcorrect nucleotide that is a cognate to the target allele. There also isthe step of (c) detecting stabilized ternary complexes at the features,thereby identifying the target alleles.

In another embodiment, a method for identifying target alleles in amixture of nucleic acids includes the step of (a) providing an array offeatures, where different allele-specific primers are attached atdifferent features of the array. There also is the step of (b)contacting the array with a plurality of nucleic acid templates,engineered DNA polymerases and nucleotides to form a plurality ofstabilized ternary complexes at a plurality of the features. Thestabilized ternary complexes each include an engineered DNA polymerase,a template nucleic acid including a target allele of a locus, anallele-specific primer of the array hybridized to the locus, and a nextcorrect nucleotide having a cognate in the locus. The 3′-end of theallele-specific primer includes a cognate nucleotide for the targetallele. There also is the step of (c) detecting stabilized ternarycomplexes at the features, thereby identifying the target alleles.

In another embodiment, a method for identifying target alleles in amixture of nucleic acids includes the step of (a) providing an array offeatures, where different locus-specific primers are attached at a firstsubset of the features of the array, and where different allele-specificprimers are attached at a second subset of the features of the array.There also is the step of (b) contacting the array with a plurality ofnucleic acid templates, engineered DNA polymerases and nucleotides toform a plurality of stabilized ternary complexes at a plurality of thefeatures. The stabilized ternary complexes at the first subset offeatures each include an engineered DNA polymerase, a template nucleicacid including a target allele of a locus, a locus-specific primer ofthe array hybridized to the locus, and a next correct nucleotide that isa cognate to the target allele. The stabilized ternary complexes at thesecond subset of features each includes an engineered DNA polymerase, atemplate nucleic acid including a target allele of a locus, anallele-specific primer of the array hybridized to the locus, and a nextcorrect nucleotide having a cognate in the locus. The 3′-end of theallele-specific primer includes a cognate nucleotide for the targetallele. There also is the step of (c) detecting stabilized ternarycomplexes at the features, thereby identifying the target alleles.

In another embodiment, a method for identifying target alleles in amixture of nucleic acids includes the step of (a) providing an array offeatures, where different template nucleic acids are attached atdifferent features of the array. There also is the step of (b)contacting the array with a plurality of primers, engineered DNApolymerases and nucleotides to form a plurality of stabilized ternarycomplexes at a plurality of the features. The stabilized ternarycomplexes at the features each include an engineered DNA polymerase, atemplate nucleic acid attached to a feature of the array and including atarget allele of a locus, a primer hybridized to the locus, and a nextcorrect nucleotide having a cognate in the locus, where either: (i) theprimer is an allele-specific primer including a 3′ nucleotide that is acognate nucleotide for the target allele, or (ii) the primer is alocus-specific primer and the next correct nucleotide hybridizes to thetarget allele. There further is the step of (c) detecting stabilizedternary complexes at the features, thereby identifying the targetalleles.

In another embodiment, a method for identifying target alleles in amixture of nucleic acids includes the step of (a) providing an array offeatures, where engineered DNA polymerases are attached at features ofthe array. There also is the step of (b) contacting the array with aplurality of primers, template nucleic acids and nucleotides to form aplurality of stabilized ternary complexes at a plurality of thefeatures. The stabilized ternary complexes at the features each includean engineered DNA polymerase that is attached at a feature of the array,a template nucleic acid including a target allele of a locus, a primerhybridized to the locus, and a next correct nucleotide having a cognatein the locus, where either: (i) the primer is an allele-specific primerincluding a 3′ nucleotide that is a cognate nucleotide for the targetallele, or (ii) the primer is a locus-specific primer and the nextcorrect nucleotide hybridizes to the target allele. There also is thestep of (c) detecting stabilized ternary complexes at the features,thereby identifying the target alleles.

Sequencing by Binding™ Methods Using Engineered Polymerases: GeneralAspects

Described herein are polymerase-based nucleic acid Sequencing ByBinding™ reactions, wherein the polymerase undergoes transitions betweenopen and closed conformations during discrete steps of the reaction. Inone step, the polymerase binds to a primed template nucleic acid to forma binary complex, also referred to herein as the pre-insertionconformation. In a subsequent step, an incoming nucleotide is bound andthe polymerase fingers close, forming a pre-chemistry conformationcomprising the polymerase, primed template nucleic acid and nucleotide;wherein the bound nucleotide has not been incorporated. This step may befollowed by a Mg²⁺- or Mn²⁺-catalyzed chemical incorporation of the nextcorrect nucleotide, wherein nucleophilic displacement of a pyrophosphate(PPi) by the 3′-hydroxyl of the primer results in phosphodiester bondformation. This is generally referred to as nucleotide “incorporation.”The polymerase returns to an open state upon the release of PPifollowing nucleotide incorporation, and translocation initiates the nextround of reaction. Certain details of the Sequencing By Binding™procedure can be found in commonly owned U.S. patent applicationsidentified by Ser. No. 14/805,381 (now published as US Pat. App. Pub.No. US 2017/0022553 A1) and 62/375,379, the entire disclosures of thesedocuments being incorporated by reference herein for all purposes.

While a ternary complex including a primed template nucleic acidmolecule having a primer with a free 3′-hydroxyl can form in the absenceof a divalent catalytic metal ion (e.g., Mg²⁺), chemical addition ofnucleotide can proceed in the presence of the divalent metal ions. Lowor deficient levels of catalytic metal ions, such as Mg²⁺ tend to leadto non-covalent (physical) sequestration of the next correct nucleotidein a tight ternary complex. This ternary complex may be referred to as astabilized or trapped ternary complex. Other methods disclosed hereinalso can be used to produce a stabilized ternary complex. In anyreaction step described above, the polymerase configuration and/orinteraction with a nucleic acid may be monitored during an examinationstep to identify the next correct base in the nucleic acid sequence.Before or after incorporation, reaction conditions can be changed todisengage the polymerase from the primed template nucleic acid, andchanged again to remove from the local environment any reagents thatinhibit polymerase binding.

Generally speaking, the Sequencing By Binding™ procedure includes an“examination” step that identifies the next template base, andoptionally a subsequent “incorporation” step that adds one or morecomplementary nucleotides to the 3′-end of the primer component of theprimed template nucleic acid. Identity of the next correct nucleotide tobe added is determined either without, or before chemical linkage ofthat nucleotide to the 3′-end of the primer through a covalent bond. Theexamination step can involve providing a primed template nucleic acid tobe used in the procedure, and contacting the primed template nucleicacid with a polymerase enzyme (e.g., a DNA polymerase) composition andone or more test nucleotides being investigated as the possible nextcorrect nucleotide. Further, there is a step that involves monitoring ormeasuring the interaction between the polymerase and the primed templatenucleic acid in the presence of the test nucleotides.

Optionally, monitoring of the interaction can take place when the primerof the primed template nucleic acid molecule includes a blocking groupthat precludes enzymatic incorporation of an incoming nucleotide intothe primer. The interaction additionally or alternatively can take placein the presence of stabilizers (e.g., non-catalytic metal ions thatinhibit incorporation), whereby the polymerase-nucleic acid interactionis stabilized in the presence of the next correct nucleotide (i.e.,stabilizers that stabilize the ternary complex). Again, the examinationstep identifies or determines the identity of the next correctnucleotide without requiring incorporation of that nucleotide. Stateddifferently, identity of the next correct nucleotide can be establishedwithout chemical incorporation of the nucleotide into the primer,whether or not the 3′-end of the primer is blocked.

Whereas methods involving a single template nucleic acid molecule may bedescribed for convenience, these methods are exemplary. The sequencingmethods provided herein readily encompass a plurality of templatenucleic acids, wherein the plurality of nucleic acids may be clonallyamplified copies of a single nucleic acid, or disparate nucleic acids,including combinations, such as populations of disparate nucleic acidsthat are clonally amplified.

The Examination Step

Generally, an examination step in a Sequencing By Binding™ procedure inaccordance with the disclosed technique typically includes the followingsub-steps: (1) providing a primed template nucleic acid molecule (i.e.,a template nucleic acid molecule hybridized with a primer thatoptionally may be blocked from extension at its 3′-end); (2) contactingthe primed template nucleic acid molecule with a reaction mixture thatincludes at least one polymerase that can be distinguished from othersused in the procedure (e.g., by virtue of including a detectable label,or by timing of delivery to a primed template nucleic acid molecule) andone nucleotide; (3) detecting the interaction of the polymerase with theprimed template nucleic acid molecule in the presence of the nucleotideand without chemical incorporation of any nucleotide into the primedtemplate nucleic acid; and (4) determining from the detected interactionthe identity of the next base in the template nucleic acid (i.e., thecomplement of the next correct nucleotide).

In one embodiment, an examination step includes: (1) serially contactinga primed template nucleic acid (where the primer strand optionally isblocked from extension at its 3′-end) with a plurality ofdistinguishably labeled polymerase-nucleotide combinations underconditions that discriminate between formation of ternary complexes andbinary complexes; (2) detecting any ternary complexes that formed as aresult of the serial contacting steps by detecting one or more of thedistinguishably labeled polymerases from the combinations used in thedifferent contacting steps; and (3) identifying the next correctnucleotide for the primed template nucleic acid as the nucleotidecomponent of the distinguishably labeled polymerase-nucleotidecombination that formed the ternary complex. While a ternary complex maybe stabilized by non-catalytic cations that inhibit nucleotideincorporation or polymerization, primers blocked at their 3′-endsprovide alternative stabilization approaches. In some embodiments, atrivalent lanthanide cation or other stabilizing agent (e.g., a divalentmetal ion that inhibits incorporation, of a trivalent metal ion thatinhibits incorporation) may be used to inhibit dissociation of thecomplex (e.g., to “lock” the ternary complex in place). Optionally, adetectably labeled polymerase is delivered to an immobilized primedtemplate nucleic acid molecule in a flow cell in combination with asingle nucleotide to assess whether or not the nucleotide is the nextcorrect nucleotide to be incorporated. Optionally, an incorporation stepfollows the examination step that identifies the next correctnucleotide.

In a different embodiment that takes advantage of single-scan imaging toprocess a population of primed template nucleic acid molecules, anexamination step includes: (1) providing the population; (2) seriallyperforming a plurality of contacting steps (e.g., four contactingsteps), one after the other, that involve contacting the population withdifferent reagent solutions, where each reagent solution contains adistinguishable polymerase (e.g., being distinguishable from the othersby virtue of a detectable label) and a different nucleotide in thepresence of a ternary complex-stabilizing agent; (3) imaging thepopulation after performance of at least two, and preferably afterperformance of all four contacting steps to detect labels associatedwith the different distinguishable polymerase compositions, therebydetermining which members of the population participate in ternarycomplexes independently containing the different polymerases; and (4)determining identities of cognate nucleotides for different members ofthe population from the imaging results. More particularly, thedetermining step optionally may involve identifying cognate nucleotidesby assessing which polymerase(s) participated in a ternary complex for aparticular member of the population. When multiple imaging stepsconveniently can be performed, imaging and detection can take placeafter each contacting step has concluded. Notably, the serial contactingsteps can be carried out in a serial fashion so that the differentpolymerase and nucleotide combinations do not mix prior to formation oftheir respective ternary complexes. Thus, the polymerase and nucleotide(as a combination, unassociated with primed template nucleic acid) fromone step should not mingle or mix with the polymerase and nucleotide (asa combination, unassociated with primed template nucleic acid) from asubsequent step. More particularly, free (i.e., non-complexed)polymerase from a prior contacting step preferably do not mingle with(i.e., are not simultaneously present with) a nucleotide type that isfirst introduced in a subsequent contacting step. Conversely, it isacceptable to mix a free (i.e., non-complexed) nucleotide type from aprior contacting step with a polymerase used in a subsequent contactingstep.

The primer of the primed template nucleic acid optionally can be eitheran extendible primer, or a primer blocked from extension at its 3′-end(e.g., by the presence of a reversible terminator moiety). The primedtemplate nucleic acid, the polymerase and the test nucleotide arecapable of forming a ternary complex when the base of the testnucleotide is complementary to the next base of the primed templatenucleic acid molecule. The primed template nucleic acid and thepolymerase are capable of forming a binary complex when the base of thetest nucleotide is not complementary to the next base of the primedtemplate nucleic acid molecule. Optionally, the contacting occurs underconditions that favor formation of the ternary complex over formation ofthe binary complex. Optionally, the conditions that favor or stabilizethe ternary complex are provided by either: (1) the presence of areversible terminator moiety on the 3′ nucleotide of the primer of theprimed template nucleic acid molecule; or (2) the presence of anon-catalytic ion (e.g., a divalent or trivalent non-catalytic metalion) that inhibits nucleotide incorporation or polymerization.Optionally, the conditions that disfavor or destabilize binary complexesare provided by the presence of one or more monovalent cations and/orglutamate anions in the reaction mixture during the examination step.Alternatively or in addition to using these conditions, a polymeraseengineered to have reduced catalytic activity or reduced propensity forbinary complex formation can be used. The determining or identifyingstep can include identifying the base of the nucleotide that iscomplementary to the next base of the primed template nucleic acid. Thiscan be accomplished by detecting the polymerase of the ternary complex(e.g., via a label attached to the polymerase), and deducing identity ofthe cognate nucleotide from that identification.

The examination step conventionally is controlled so that nucleotideincorporation is attenuated. This being the case, a separateincorporation step (discussed elsewhere herein in greater detail) may beperformed. The separate incorporation step may be accomplished withoutthe need for monitoring, as the base has already been identified duringthe examination step. However if desired, subsequent incorporation canbe detected, for example by incorporating nucleotides having exogenouslabels. Detection at both binding and incorporation steps can providefor error checking and increased sequencing accuracy. A reversiblyterminated nucleotide (whether labeled or not) may be used in theincorporation step to prevent the addition of more than one nucleotideduring a single cycle.

The Sequencing By Binding™ method allows for controlled determination ofa template nucleic acid base (e.g., by identifying a next correctnucleotide) without the need for labeled nucleotides, as the interactionbetween the polymerase and template nucleic acid can be monitoredwithout a label on the nucleotide. Template nucleic acid molecules maybe sequenced under examination conditions which do not requireattachment of template nucleic acid or polymerase to a solid support.However, in certain preferred embodiments, primed template nucleic acidsto be sequenced are attached to a solid support, such as an interiorsurface of a flow cell. The compositions, methods and systems describedherein provide numerous advantages over previous systems, such ascontrolled reaction conditions, unambiguous determination of sequence,long read lengths, low overall cost of reagents, and low instrumentcost. Accordingly, in some embodiments, a polymerase having a sequenceset forth herein can form a stabilized ternary complex on a solidsupport via binding to a primed template nucleic acid that is attachedto the solid support.

Alternatively or in addition to attaching primed template nucleic acidsto a solid support, one or more polymerase molecules can be attached tothe solid support. Attachment of polymerase to a solid support canprovide an advantage in localizing the polymerase for a subsequentdetection step. This can be useful for example, when screeningpolymerase variants for ability to form a stabilized ternary complexwith a primed template nucleic acid and nucleotide that are deliveredvia solution phase. Alternatively, attachment of the polymerase can beuseful for localizing the polymerase at a feature where a particularnucleic acid resides.

Optionally, the provided methods further include a wash step. The washstep can occur before or after any other step in the method. Optionally,the wash step is performed after each of the serially contacting steps,wherein the primed template nucleic acid molecule is contacted with oneof the distinguishably labeled polymerase-nucleotide combinations.Optionally, the wash step is performed prior to the monitoring stepand/or prior to the determining or identifying step. Optionally, thewash step occurs under conditions that stabilize the ternary complex.Optionally, the conditions result from the presence of a reversibleterminator moiety on the 3′ nucleotide of the primer of the primedtemplate nucleic acid molecule. Optionally, the conditions include astabilizing agent. Optionally, the stabilizing agent is a non-catalyticmetal ion (e.g., a divalent non-catalytic metal ion) that inhibitsnucleotide incorporation or polymerization. Non-catalytic metal ionsinclude, but are not limited to, calcium, strontium, scandium, titanium,vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium,germanium, arsenic, selenium, rhodium, europium, and terbium ions.Optionally, the wash buffer includes nucleotides from previouscontacting steps, but does not include the distinguishably labeledpolymerase composition of a prior polymerase-nucleotide combination.Including the nucleotides from previous contacting steps can provide theadvantage of stabilizing previously formed ternary complexes fromunwanted disassociation. This in turn prevents unwanted loss of signaldue to washing away previously formed ternary complexes or emergence oferroneous signals due to reconstitution between one or more component(s)of previously formed ternary complexes and one or more component(s) ofan incoming reagent. Optionally, the ternary complex has a half-life andthe wash step is performed for a duration shorter than the half-life ofthe ternary complex formed when a nucleotide molecule provides a basethat is complementary to the next base of the primed template nucleicacid molecule.

The examination step may be controlled, in part, by providing reactionconditions to prevent chemical incorporation of a nucleotide, whileallowing determination of the identity of the next correct base on theprimed template nucleic acid molecule. Such reaction conditions may bereferred to as examination reaction conditions. Optionally, a ternarycomplex is formed under examination conditions.

Optionally, the examination conditions accentuate the difference inaffinity for polymerase to primed template nucleic acids in the presenceof different nucleotides, for example by destabilizing binary complexes.Optionally, the examination conditions cause differential affinity ofthe polymerase for the primed template nucleic acid in the presence ofdifferent nucleotides. By way of example, the examination conditionsthat cause differential affinity of the polymerase for the primedtemplate nucleic acid in the presence of different nucleotides include,but are not limited to, high salt and glutamate ions. For example, thesalt may dissolve in aqueous solution to yield a monovalent cation, suchas a monovalent metal cation (e.g., sodium ion or potassium ion).Optionally, the salt that provides the monovalent cations (e.g.,monovalent metal cations) further provides glutamate anions. Optionally,the source of glutamate ions can be potassium glutamate. In someinstances, the concentrations of potassium glutamate that can be used toalter polymerase affinity of the primed template nucleic acid extendfrom 10 mM to 1.6 M of potassium glutamate, or any amount in between 10mM and 1.6 M. As indicated above, high salt refers to a concentration ofsalt from 50 to 1,500 mM salt.

Optionally, examination involves detecting polymerase interaction with atemplate nucleic acid where the interaction of one or more polymerasecompositions (e.g., where each different polymerase composition containsa different polymerase, or a different combination of two or morepolymerases) can be distinguished. Detection may include optical,electrical, thermal, acoustic, chemical and mechanical means.Optionally, examination is performed after a wash step, wherein the washstep removes any non-bound reagents (e.g., unbound polymerases and/ornucleotides) from the region of observation. This may occur at the endof a series of steps involving contacting of a primed template nucleicacid molecule with a plurality of distinguishable polymerase-nucleotidecombinations. Optionally, examination is performed during a wash step,such that the dissociation kinetics of the polymerase-nucleic acid orpolymerase-nucleic acid-nucleotide complexes may be monitored and usedto determine the identity of the next base. Optionally, examination isperformed during the course of addition of the examination reactionmixture (or first reaction mixture), such that the association kineticsof the polymerase to the nucleic acid may be monitored and used todetermine the identity of the next base on the nucleic acid. Optionally,examination involves distinguishing ternary complexes from binarycomplexes of polymerase and nucleic acid. Optionally, examination isperformed under equilibrium conditions where the affinities measured areequilibrium affinities. Multiple examination steps comprising differentor similar examination reagents, may be performed sequentially toascertain the identity of the next template base. Multiple examinationsteps may be utilized in cases where multiple template nucleic acids arebeing sequenced simultaneously in one sequencing reaction, whereindifferent nucleic acids react differently to the different examinationreagents. Optionally, multiple examination steps may improve theaccuracy of next base determination. Optionally, a single examinationstep is used to identify the next correct nucleotide, out of a pluralityof possible nucleotides (e.g., four possible nucleotides), for differentprimed template nucleic acid molecules among a population.

Generally, the examination step involves binding a polymerase to thepolymerization initiation site of a primed template nucleic acid in areaction mixture comprising one or more nucleotides, and monitoring theinteraction. In certain preferred embodiments, this is accomplishedusing only a single polymerase in combination with one or morenucleotides. This may involve use of only a single nucleotide.Optionally, a nucleotide is sequestered within the polymerase-primedtemplate nucleic acid complex to form a ternary complex under conditionsin which incorporation of the enclosed nucleotide by the polymerase isattenuated or inhibited. Optionally, the ternary complex isalternatively or additionally stabilized by the presence of a blockingmoiety (e.g., a reversible terminator moiety) on the 3′ terminalnucleotide of the primer. Optionally a stabilizer is added to stabilizethe ternary complex in the presence of the next correct nucleotide. Thisternary complex is in a stabilized or polymerase-trapped pre-chemistryconformation.

Contacting Steps

In the provided methods, contacting of the primed template nucleic acidmolecule with a reaction mixture that includes a polymerase compositionand one nucleotide optionally occurs under conditions that stabilizeformation of the ternary complex, and that destabilize formation ofbinary complexes. These conditions can be provided by alternativeapproaches that are a matter of choice by the end-user.

Optionally, the conditions comprise contacting the primed templatenucleic acid molecule with a buffer that regulates osmotic pressure.Optionally, the reaction mixture used in the examination step includesthe buffer that regulates osmotic pressure. Optionally, the buffer is ahigh salt buffer that includes a monovalent ion, such as a monovalentmetal ion (e.g., potassium ion or sodium ion) at a concentration of from50 to 1,500 mM. Salt concentrations in the range of from 100 to 1,500mM, and from 200 to 1,500 mM also are highly preferred. Optionally, thebuffer further includes a source of glutamate ions (e.g., potassiumglutamate). Optionally, the conditions that stabilize formation of theternary complex involve contacting the primed template nucleic acidmolecule with a stabilizing agent. Optionally, the reaction mixture usedduring the examination step includes a stabilizing agent. Optionally,the stabilizing agent is a non-catalytic metal ion (e.g., a divalent ortrivalent non-catalytic metal ion). Non-catalytic metal ions useful inthis context include, but are not limited to, calcium, strontium,scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper,zinc, gallium, germanium, arsenic, selenium, rhodium, europium, andterbium.

Optionally, the contacting step is facilitated by the use of a flow cellor chamber, multiwell plate, etc. Flowing liquid reagents through theflow cell, which contains an interior solid support surface (e.g., aplanar surface), conveniently permits reagent exchange or replacement.Immobilized to the interior surface of the flow cell is one or moreprimed template nucleic acids to be sequenced or interrogated using theprocedures described herein. Typical flow cells will includemicrofluidic valving that permits delivery of liquid reagents (e.g.,components of the “reaction mixtures” discussed herein) to an entryport. Liquid reagents can be removed from the flow cell by exitingthrough an exit port.

As discussed above, in certain embodiments it is desirable to avoidmixing one distinguishably labeled polymerase-nucleotide combinationreagent with a subsequent polymerase-nucleotide combination reagentduring the plurality of serial contacting steps. This can beaccomplished by including an intervening wash step between each of theserial contacting steps. This may be done by alternatively flowing abinding mixture that includes a polymerase-nucleotide combinationreagent and a wash solution through a flow cell. By another approach,robotic fluid handling may be used to perform reagent exchanges whenusing a multiwell formatted platform.

Detecting Steps

Detecting (e.g., via monitoring or measuring) the interaction of apolymerase with a primed template nucleic acid molecule in the presenceof a nucleotide molecule may be accomplished in many different ways. Forexample, monitoring can include measuring association kinetics for theinteraction between the primed template nucleic acid, the polymerase,and any one of the four nucleotide molecules. Monitoring the interactionof the polymerase with the primed template nucleic acid molecule in thepresence of a nucleotide molecule can include measuring equilibriumbinding constants between the polymerase and primed template nucleicacid molecule (i.e., equilibrium binding constants of polymerase to thetemplate nucleic acid in the presence of any one or the fournucleotides). Thus, for example, the monitoring includes measuring theequilibrium binding constant of the polymerase to the primed templatenucleic acid in the presence of any one of the four nucleotides.Monitoring the interaction of the polymerase with the primed templatenucleic acid molecule in the presence of a nucleotide molecule includes,for example, measuring dissociation kinetics of the polymerase from theprimed template nucleic acid in the presence of any one of the fournucleotides. Optionally, monitoring the interaction of the polymerasewith the primed template nucleic acid molecule in the presence of anucleotide molecule includes measuring dissociation kinetics of thedissociation of the closed-complex (i.e., dissociation of the primedtemplate nucleic acid, the polymerase, and any one of the fournucleotide molecules). Optionally, the measured association kinetics aredifferent depending on the identity of the nucleotide molecule.Optionally, the polymerase has a different affinity for each of the fourtypes of nucleotide molecules. Optionally, the polymerase has adifferent dissociation constant for each of the four types of nucleotidemolecules in each type of ternary complex. Association, equilibrium anddissociation kinetics are known and can be readily determined by one inthe art. See, for example, Markiewicz et al., Nucleic Acids Research40(16):7975-84 (2012); Xia et al., J. Am. Chem. Soc. 135(1):193-202(2013); Brown et al., J. Nucleic Acids, Article ID 871939, 11 pages(2010); Washington, et al., Mol. Cell. Biol. 24(2):936-43 (2004); Walshand Beuning, J. Nucleic Acids, Article ID 530963, 17 pages (2012); andRoettger, et al., Biochemistry 47(37):9718-9727 (2008), which areincorporated by reference herein in their entireties.

The detecting step can include monitoring the steady state interactionof the polymerase with the primed template nucleic acid molecule in thepresence of the first nucleotide molecule, without chemicalincorporation of the first nucleotide molecule into the primer of theprimed template nucleic acid molecule. Optionally, the detectingincludes monitoring dissociation of the polymerase with the primedtemplate nucleic acid molecule in the presence of the first nucleotidemolecule, without chemical incorporation of the first nucleotidemolecule into the primer of the primed template nucleic acid molecule.Optionally, the detecting includes monitoring association of thepolymerase with the primed template nucleic acid molecule in thepresence of the first nucleotide molecule, without chemicalincorporation of the first nucleotide molecule into the primer of theprimed template nucleic acid molecule. Again, the test nucleotides inthese procedures may be native nucleotides (i.e., unlabeled), labelednucleotides (e.g., including fluorescent or Raman scattering labels), ornucleotide analogs (e.g., nucleotides modified to include reversibleterminator moieties with or without detectable label moieties). It willbe understood that a detection technique can accumulate signal over arelatively brief duration as is typically understood to be a singletimepoint acquisition or, alternatively, signal can be continuouslymonitored over time as is typical of a time-based acquisition. It isalso possible to acquire a series of timepoints to obtain a time-basedacquisition.

In the sequencing methods provided herein, either a chemical block onthe 3′ nucleotide of the primer of the primed template nucleic acidmolecule (e.g., a reversible terminator moiety on the base or sugar ofthe nucleotide), the absence of a catalytic metal ion in the reactionmixture, or the absence of a catalytic metal ion in the active site ofthe polymerase prevents the chemical incorporation of the nucleotideinto the primer of the primed template nucleic acid. Optionally, thechelation of a catalytic metal ion in the reaction mixtures of thecontacting step prevents the chemical incorporation of the nucleotideinto the primer of the primed template nucleic acid. Optionally, anon-catalytic metal ion acts as a stabilizer for the ternary complex inthe presence of the next correct nucleotide. Optionally, thesubstitution of a catalytic metal ion in the reaction mixtures of thecontacting step with a non-catalytic metal ion prevents the chemicalincorporation of the nucleotide molecule to the primed template nucleicacid. Optionally, the catalytic metal ion is magnesium. The metal ionmechanisms of polymerases postulate that a low concentration of metalions may be needed to stabilize the polymerase-nucleotide-DNA bindinginteraction. See, for instance, Section 27.2.2, Berg J M, Tymoczko J L,Stryer L, Biochemistry 5th Edition, WH Freeman Press, 2002.

Optionally, a low concentration of a catalytic ion in the reactionmixture used during the examination step prevents the chemicalincorporation of the nucleotide molecule to the primed template nucleicacid. Optionally, a low concentration is from about 1 μM to about 100μM. Optionally, a low concentration is from about 0.5 μM to about 5 μM.Optionally, the reaction mixture used during the examination stepincludes cobalt ions and the incorporating step involves contacting withan incorporation reaction mixture that includes a higher concentrationof cobalt ions as compared to the concentration of cobalt ions in thefirst reaction mixture.

In an exemplary sequencing reaction, the examination step involvesformation and/or stabilization of a ternary complex including apolymerase, primed template nucleic acid, and nucleotide.Characteristics of the formation and/or release of the ternary complexcan be detected to identify the enclosed nucleotide and therefore thenext base in the template nucleic acid. Ternary complex characteristicscan be dependent on the sequencing reaction components (e.g.,polymerase, primer, template nucleic acid, nucleotide) and/or reactionmixture components and/or conditions.

The examination step involves detecting the interaction of a polymerasewith a template nucleic acid in the presence of a nucleotide. Theformation of a ternary complex may be detected or monitored. Optionally,the absence of formation of ternary complex is detected or monitored.Optionally, the dissociation of a ternary complex is monitored.Optionally, the incorporation step involves detecting or monitoringincorporation of a nucleotide. Optionally, the incorporation stepinvolves detecting or monitoring the absence of nucleotideincorporation.

Any process of the examination and/or incorporation step may be detectedor monitored. Optionally, a polymerase has a detectable tag (e.g., afluorescent label or a Raman scattering tag). Optionally, the detectabletag or label on the polymerase is removable. Generally speaking, whenusing single-scan imaging, among the series of distinguishablepolymerase and nucleotide combinations employed in the procedure, as fewas two polymerases among the plurality of differentpolymerase-nucleotide combinations will harbor detectable labels. Asindicated elsewhere herein, this can provide information about fourdifferent nucleotides based on monitoring ternary complex formation. Asingle polymerase label can be used when multiple scans (e.g., fourindependent scans) are performed.

Optionally, a nucleotide of a particular type (e.g., dATP, dCTP, dGTP,dTTP, or analogs thereof) is made available to a polymerase in thepresence of a primed template nucleic acid molecule. The reaction isdetected or monitored, wherein, if the nucleotide is a next correctnucleotide, the polymerase may be stabilized to form a ternary complex.If the nucleotide is an incorrect nucleotide, a ternary complex maystill be formed; however, without the additional assistance ofstabilizing agents or reaction conditions (e.g., absence of catalyticions, polymerase inhibitors, salt), the ternary complex may dissociate.The rate of dissociation is dependent on the affinity of the particularcombination of polymerase, template nucleic acid, and nucleotide, aswell as reaction conditions. Optionally, the affinity is measured as anoff-rate. Optionally, the affinity is different between differentnucleotides for the ternary complex. For example, if the next base inthe template nucleic acid downstream of the 3′-end of the primer is G,the polymerase-nucleic acid affinity, measured as an off-rate, isexpected to be different based on whether dATP, dCTP, dGTP or dTTP (oranalogs thereof) are added. In this case, dCTP would have the slowestoff-rate, with the other nucleotides providing different off-rates forthe interaction. Optionally, the off-rate may be different depending onthe reaction conditions, for example, the presence of stabilizing agents(e.g., absence of magnesium or inhibitory compounds) or reactionconditions (e.g., nucleotide modifications or modified polymerases).

Once the identity of the next correct nucleotide is determined, 1, 2, 3,4 or more nucleotide types may be introduced simultaneously to thereaction mixture under conditions that specifically target the formationof a ternary complex. Excess nucleotides optionally may be removed fromthe reaction mixture and the reaction conditions modulated toincorporate the next correct nucleotide of the ternary complex. Thissequencing reaction ensures that only one nucleotide is incorporated persequencing cycle. Preferably, reversible terminator nucleotides areemployed in the incorporation step, and optionally, the reversibleterminator nucleotides are not labeled with fluorescent or other labels.

Identifying Steps

The identity of the next correct base or nucleotide can be determined bydetecting the presence, formation, and/or dissociation of the ternarycomplex. The identity of the next correct nucleotide may be determinedwithout covalently incorporating the nucleotide into to the primer atits 3′-end. Optionally, the identity of the next base is determined bydetecting the affinity of the polymerase and the primed template nucleicacid in the presence of added nucleotides. Optionally, the affinity ofthe polymerase and the primed template nucleic acid in the presence ofthe next correct nucleotide may be used to determine the next correctbase on the template nucleic acid. Optionally, the affinity of thepolymerase to the primed template nucleic acid in the presence of anincorrect nucleotide may be used to determine the next correct base onthe template nucleic acid.

In certain embodiments, a ternary complex that includes a primedtemplate nucleic acid (or a blocked primed template nucleic acid) isformed in the presence of a polymerase and a plurality of nucleotides.Cognate nucleotide participating in the ternary complex optionally isidentified by observing destabilization of the complex that occurs whenthe cognate nucleotide is absent from the reaction mixture. This isconveniently carried out, for example, by exchanging one reactionmixture for another. Here, loss of the complex is an indicator ofcognate nucleotide identity. Loss of binding signal (e.g., a fluorescentbinding signal associated with a particular locus on a solid support)can occur when the primed template nucleic acid is exposed to a reactionmixture that does not include the cognate nucleotide. Optionally,maintenance of a ternary complex in the presence of a single nucleotidein a reaction mixture also can indicate identity of the cognatenucleotide. When reversible terminator nucleotides are employed, removalof excess nucleotides is unnecessary because only a single reversibleterminator nucleotide can be incorporated before the reversibleterminator moiety is removed.

Incorporation Steps

Optionally, incorporation proceeds after the cognate nucleotide has beenidentified in an examination procedure using a first polymerase inaccordance with the disclosure. Incorporation optionally may employ apolymerase different from the one used in the examination step, togetherwith a nucleotide analog. For example, the nucleotide analog can be anunlabeled reversible terminator nucleotide corresponding to theidentified cognate nucleotide (i.e., the reversible terminatornucleotide and the cognate nucleotide are both complementary to the samebase of the template strand). Also significantly, cognate nucleotidesfor a plurality of different primed template nucleic acids havingdifferent sequences advantageously can be identified using only a singleimaging step. This is sometimes referred to as “single-scan imaging.”Thus, the provided approach is both simple to implement and rapid toanalyze.

The methods described herein optionally include an incorporation step.The incorporation step involves covalently incorporating one or morenucleotides at the 3′-end of a primer hybridized to a template nucleicacid. In a preferred embodiment, only a single nucleotide isincorporated at the 3′-end of the primer. Optionally, multiplenucleotides of the same kind are incorporated at the 3′-end of theprimer. Optionally, multiple nucleotides of different kinds areincorporated at the 3′-end of the primer. Incorporated nucleotidesalternatively can be unlabeled nucleotides, reversible terminatornucleotides, or detectably labeled nucleotide analogs. The polymerasecan dissociate from the polymerization initiation site after nucleotideincorporation or can be retained at the polymerization initiation siteafter incorporation.

The incorporation reaction may be facilitated by an incorporationreaction mixture. Optionally, the incorporation reaction mixture has adifferent composition of nucleotides than the examination reaction. Forexample, the examination reaction can include one type of nucleotide andthe incorporation reaction can include another type of nucleotide.Optionally, the incorporation reaction includes a polymerase that isdifferent from the polymerases of the examination step. By way ofanother example, the examination reaction comprises one type ofnucleotide and the incorporation reaction comprises four types ofnucleotides, or vice versa. In yet another example, the examinationreaction uses four different reagents, each containing one of four typesof nucleotides, such that the four types of nucleotides are sequentiallypresent, and the incorporation reaction can include the four types ofnucleotides in a simultaneous mixture. As a further example, a firstexamination reaction can introduce a first type of nucleotide, a secondexamination reaction can introduce a second type of nucleotide alongwith the first type of nucleotide, a third examination reaction canintroduce a third type of nucleotide along with the first and secondtypes of nucleotides, a fourth examination reaction can introduce afourth type of nucleotide along with the first, second and third typesof nucleotides, and the incorporation reaction can include the first,second, third and fourth types of nucleotides in a simultaneous mixture.Optionally, an examination reaction mixture is altered or replaced by anincorporation reaction mixture. Optionally, an incorporation reactionmixture includes a catalytic metal ion (e.g., a divalent catalytic metalion), a monovalent metal cation (e.g., potassium ions or sodium ions),glutamate ions, or a combination thereof.

There is flexibility in the nature of the nucleotide used in theincorporation step. For example, the at least one nucleotide can includea 3′-oxygen, which can be, for example, a member of a free 3′-hydroxylgroup. Optionally, the 3′ position of the at least one nucleotidemolecule is modified to include a 3′ terminator moiety. The 3′terminator moiety may be a reversible terminator or may be anirreversible terminator. Optionally, the reversible terminatornucleotide includes a 3′-ONH₂ moiety attached at the 3′ position of thesugar moiety. Optionally, the reversible terminator of the at least onenucleotide molecule is replaced or removed before or after theexamination step. Further examples of useful reversible terminatormoieties are described, for example, in Bentley et al., Nature 456:53-59(2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO07/123744; U.S. Pat. No. 7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat.No. 7,315,019; U.S. Pat. No. 7,405,281, and US 2008/0108082, each ofwhich is incorporated herein by reference

Nucleotides (e.g., incorporable nucleotides that are neither reversibleterminator nucleotides, nor irreversible terminator nucleotides) presentin the reaction mixture but not sequestered in a ternary complex maycause multiple nucleotide insertions during an incorporation reaction. Awash step can be employed prior to the chemical incorporation step topromote or ensure only the nucleotide sequestered within a trappedternary complex being available for incorporation during theincorporation step. Optionally, free nucleotides may be removed byenzymes such as phosphatases. The trapped polymerase complex may be aternary complex, a stabilized ternary complex or ternary complexinvolving the polymerase, primed template nucleic acid and next correctnucleotide.

Optionally, the nucleotide enclosed within the ternary complex of theexamination step is incorporated into the 3′-end of the template nucleicacid primer during the incorporation step. For example, a stabilizedternary complex of the examination step includes an incorporated nextcorrect nucleotide.

Optionally, the incorporation step involves replacing a nucleotide fromthe examination step and incorporating another nucleotide into the3′-end of the template nucleic acid primer. The incorporation step caninvolve releasing a nucleotide from within a ternary complex (e.g., thenucleotide is a modified nucleotide or nucleotide analog) andincorporating a nucleotide of a different kind into the 3′-end of theprimer of the primed template nucleic acid molecule. Optionally, thereleased nucleotide is removed and replaced with an incorporationreaction mixture containing a next correct nucleotide. For example, theincorporated nucleotide can be a reversible terminator nucleotide, suchas an unlabeled reversible terminator nucleotide that does not include adetectable fluorophore.

Suitable reaction conditions for incorporation may involve replacing theexamination reaction mixture with an incorporation reaction mixture.Optionally, nucleotide(s) present in the examination reaction mixtureare replaced with one or more nucleotides in the incorporation reactionmixture. Optionally, the polymerase(s) present during the examinationstep is replaced during the incorporation step. By this approach it ispossible to employ different types of polymerase in the examination andincorporation steps. Optionally, the polymerase present during theexamination step is modified during the incorporation step. Optionally,the one or more nucleotides present during the examination step aremodified during the incorporation step. The reaction mixture and/orreaction conditions present during the examination step may be alteredby any means during the incorporation step. These means include, but arenot limited to, removing reagents, chelating reagents, dilutingreagents, adding reagents, altering reaction conditions such asconductivity or pH, and any combination thereof.

Optionally, the provided reaction mixture(s), including theincorporation reaction mixture(s), include at least one nucleotidemolecule that is a non-incorporable nucleotide or a nucleotide incapableof incorporation into the nucleic acid strand. In other words, theprovided reaction mixture(s) can include one or more nucleotidemolecules incapable of incorporation into the primer of the primedtemplate nucleic acid molecule. Such nucleotides incapable ofincorporation include, for example, monophosphate and diphosphatenucleotides. In another example, the nucleotide may containmodification(s) to the triphosphate group that make the nucleotidenon-incorporable. Examples of non-incorporable nucleotides may be foundin U.S. Pat. No. 7,482,120, which is incorporated by reference herein inits entirety. Optionally, the primer may not contain a free hydroxylgroup at its 3′-end, thereby rendering the primer incapable ofincorporating any nucleotide, and, thus, making any nucleotidenon-incorporable.

A polymerase inhibitor optionally may be included with the reactionmixtures containing test nucleotides in the examination step to trap thepolymerase on the nucleic acid upon binding the next correct nucleotide.Optionally, the polymerase inhibitor is a pyrophosphate analog.Optionally, the polymerase inhibitor is an allosteric inhibitor.Optionally, the polymerase inhibitor is a DNA or an RNA aptamer.Optionally, the polymerase inhibitor competes with a catalyticion-binding site in the polymerase. Optionally, the polymerase inhibitoris a reverse transcriptase inhibitor. The polymerase inhibitor may be anHIV-1 reverse transcriptase inhibitor or an HIV-2 reverse transcriptaseinhibitor. The HIV-1 reverse transcriptase inhibitor may be a(4/6-halogen/MeO/EtO-substituted benzo[d]thiazol-2-yl)thiazolidin-4-one.

The provided method may further include preparing the primed templatenucleic acid molecule for a next examination step after theincorporation step. Optionally, the preparing includes subjecting theprimed template nucleic acid or the nucleic acid/polymerase complex toone or more wash steps; a temperature change; a mechanical vibration; apH change; a chemical treatment to remove reversible terminatormoieties; or an optical stimulation. Optionally, the wash step comprisescontacting the primed template nucleic acid or the primed templatenucleic acid/polymerase complex with one of more buffers, detergents,protein denaturants, proteases, oxidizing agents, reducing agents, orother agents capable of releasing internal crosslinks within apolymerase or crosslinks between a polymerase and nucleic acid.

In some embodiments, the disclosed techniques do not share restrictionson detectable labels that characterize certain other techniques used inthe DNA sequencing field. For example, in some embodiments, there is norequirement for a label (e.g., a FRET partner) to be present on thepolymerase, the primed template nucleic acid, or the nucleotidesequestered within a ternary complex. Alternatively, FRET partner can bepresent on a polymerase having a sequence set forth herein. The FRETpartner can be positioned to interact with a FRET partner on a primer,template or nucleotide. The FRET partner that is attached to thepolymerase can be a donor or acceptor in a FRET interaction.

In certain embodiments the polymerase is unlabeled, or does not generateany signal used for identifying cognate or non-cognate nucleotide. Inother embodiments, the polymerase includes a covalently attacheddetectable label, such as a fluorescent label, a Raman scattering tag,etc. The polymerase preferably does not transfer energy to any labelednucleotide to render it detectable by the detection apparatus used forcarrying out the technique. The label or dye of the detectablenucleotide(s) or polymerase(s) employed in the procedure preferably isnot an intercalating dye (e.g., as disclosed in U.S. Pat. No.8,399,196), that changes its signal-generating properties (e.g.,fluorescent output) upon binding DNA. As well, the label or dye presenton the labeled nucleotide need not be a conformationally sensitive dyethat changes spectral properties when it is the cognate nucleotidepresent in a ternary complex.

In the provided sequencing methods, the next correct nucleotide can beidentified before an incorporation step, thereby allowing theincorporation step to avoid the use of labeled reagents and/ormonitoring. Optionally, nucleotides used for identifying the nextcorrect nucleotide are free of attached detectable tags or labels.Indeed, in some preferred embodiments, none of the nucleotides in theprocedure contains a detectable label. Optionally, a nucleotide includesa detectable label, but the label is not detected in the method ofidentifying the next correct nucleotide. Optionally, when fluorescentlylabeled nucleotides are used for determining identity of the nextcorrect nucleotide, the fluorescent label shows substantially no changein its fluorescent properties (excitation and emission) as the result ofinteraction with any nucleotide (e.g., through base pairing in a ternarycomplex), or as the result of a conformational change to the polymeraseitself. Thus, for example, polymerase signaling does not require energytransfer to or from the detectable label because of nucleotideinteraction with the polymerase. Optionally, the detectable label of adistinguishably labeled polymerase is a fluorescent label, but thefluorescent label is not an intercalating dye that changes propertiesupon binding a primed template nucleic acid molecule.

In certain preferred embodiments, the polymerase is labeled with afluorescent detectable label, where the detectable label showssubstantially no change in its fluorescent properties (excitation andemission) as the result of interaction with any nucleotide, or as theresult of a conformational change to the polymerase itself. Thus, forexample, labeled polymerase signaling does not require energy transferto or from the detectable label because of nucleotide interaction withthe polymerase. Optionally, the detectable label of a distinguishablylabeled polymerase is a fluorescent label, but the fluorescent label isnot an intercalating dye that changes properties upon binding a primedtemplate nucleic acid molecule. Optionally, a polymerase having asequence set forth herein can be attached to a nucleic acidintercalating dye. Exemplary intercalating dyes and methods for theiruse are set forth, for example, in U.S. Pat. No. 8,399,196, which isincorporated herein by reference.

The examination step of the sequencing reaction may be repeated 1, 2, 3,4 or more times prior to the incorporation step. The examination andincorporation steps may be repeated for a predefined number of cycles,until the desired sequence of the template nucleic acid is obtained oruntil certain reaction criteria are reached such as a minimum signalintensity or signal to noise ratio.

Sequencing Methods Employing Destabilization of Ternary ComplexesContaining Engineered Polymerases: General Aspects

Engineered DNA polymerases in accordance with this disclosure can beused in sequencing procedures employing ternary complex destabilization,for example as described in commonly owned U.S. patent applicationidentified by Ser. No. 15/581,828, published as US 2017/0314064 A1, theentire disclosure of which is incorporated by reference herein. Thetechnique involves initial formation of a ternary complex using aplurality of nucleotides, and then subsequently investigating stabilityof the complex under a series of changed reagent conditions. Thesechanged conditions involve progressive removal of nucleotides from acontrolled series of binding reaction mixtures. For example, a ternarycomplex that includes a particular dNTP will require that dNTP in afirst reagent solution to maintain integrity of the complex. Exchangingthe first reagent solution with a second reagent solution that does notinclude the critical dNTP will cause destabilization of the complex,which can be detected as an indicator of nucleotide identity.

In one embodiment, there is a method of identifying a nucleotide thatincludes a base complementary to the next base of a template strandimmediately downstream of a primer in a primed template nucleic acidmolecule. The method can begin with the step of (a) providing a blockedprimed template nucleic acid molecule including a reversible terminatormoiety that precludes the 3′-terminus of the blocked primed templatenucleic acid molecule from participating in phosphodiester bondformation. There also is the step of (b) contacting the blocked primedtemplate nucleic acid molecule with a first reaction mixture thatincludes an engineered DNA polymerase, and a plurality of differentnucleotide molecules. As a result, there forms a stabilized ternarycomplex that includes one of the plurality of different nucleotidemolecules. There also is the step of (c) contacting the stabilizedternary complex with a second reaction mixture that includes at leastone of the different nucleotide molecules and that does not include afirst nucleotide molecule of the plurality of different nucleotidemolecules. There also is the step of (d) monitoring interaction of thepolymerase and the blocked primed template nucleic acid molecule incontact with the second reaction mixture to detect any of the stabilizedternary complex remaining after step (c). Still further, there is thestep of (e) identifying the nucleotide that includes the basecomplementary to the next base of the template strand using results fromstep (d).

In another embodiment, there is a method of identifying a nucleotidethat includes a base complementary to the next base of a template strandimmediately downstream of a primer in a primed template nucleic acidmolecule. The method can begin with the step of (a) providing the primedtemplate nucleic acid molecule. There also is the step of (b) contactingthe primed template nucleic acid molecule with a first reaction mixturethat includes an engineered DNA polymerase and a plurality of differentnucleotide molecules. As a result, there forms a stabilized ternarycomplex that includes one of the plurality of different nucleotidemolecules. There also is the step of (c) contacting the primed templatenucleic acid molecule, after step (b), with a second reaction mixturethat includes at least one of the different nucleotide molecules andthat does not include a first nucleotide molecule of the plurality ofdifferent nucleotide molecules. There also is the step of (d) monitoringinteraction of the engineered DNA polymerase and the primed templatenucleic acid molecule in the second reaction mixture, withoutincorporating any nucleotide into the primer, to detect any of thestabilized ternary complex remaining after step (c). Still further,there is the step of (e) identifying the nucleotide that includes thebase complementary to the next base of the template strand using resultsfrom step (d).

In another embodiment, there is a method of identifying a nucleotidethat includes a base complementary to the next base of a template strandimmediately downstream of a primer in a primed template nucleic acidmolecule. The method can begin with the step of (a) providing the primedtemplate nucleic acid molecule. There also is the step of (b) contactingthe primed template nucleic acid molecule with a first reaction mixturethat includes an engineered DNA polymerase, but does not include anynucleotide, whereby a binary complex forms. There also is the step of(c) contacting the binary complex with a second reaction mixture thatincludes a plurality of different nucleotide molecules, whereby astabilized ternary complex forms if one of the plurality of differentnucleotide molecules includes the base complementary to the next base ofthe template strand. There also is the step of (d) detecting, withoutincorporating any nucleotide into the primer, any of the stabilizedternary complex that may have formed. There also is the step of (e)contacting the primed template nucleic acid molecule, after step (d),with a third reaction mixture that includes at least one of thedifferent nucleotide molecules and that does not include a firstnucleotide molecule of the plurality of different nucleotide molecules.There also is the step of (f) detecting, without incorporating anynucleotide into the primer, any of the stabilized ternary complexremaining after step (e). Still further, there is the step of (g)identifying the nucleotide that includes the base complementary to thenext base of the template strand using results from both of detectingsteps (d) and (f).

Reaction Mixtures

Nucleic acid sequencing reaction mixtures, or simply “reactionmixtures,” can include one or more reagents that are commonly present inpolymerase-based nucleic acid synthesis reactions. Reaction mixturereagents include, but are not limited to, enzymes (e.g., polymerase(s)),dNTPs (or analogs thereof), template nucleic acids, primer nucleic acids(including 3′ blocked primers), salts, buffers, small molecules,co-factors, metals, and ions. The ions may be catalytic ions, divalentcatalytic ions, non-catalytic ions, non-covalent metal ions, or acombination thereof. The reaction mixture can include salts, such asNaCl, KCl, potassium acetate, ammonium acetate, potassium glutamate,NH₄Cl, or (NH₄HSO₄), that ionize in aqueous solution to yield monovalentcations. The reaction mixture can include a source of ions, such as Mg²⁺or Mn²⁺, Co²⁺, Cd²⁺ or Ba²⁺ ions. The reaction mixture can include tin,Ca²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺ (e.g., Fe(II)SO₄), or Ni²⁺, or otherdivalent or trivalent non-catalytic metal cation that stabilizes ternarycomplexes by inhibiting formation of phosphodiester bonds between theprimed template nucleic acid molecule and the cognate nucleotide.

The buffer can include Tris, Tricine, HEPES, MOPS, ACES, MES,phosphate-based buffers, and acetate-based buffers. The reaction mixturecan include chelating agents such as EDTA, EGTA, and the like.Optionally, the reaction mixture includes cross-linking reagents.Provided herein are first reaction mixtures, optionally, used during theexamination step, as well as incorporation reaction mixtures used duringnucleotide incorporation that can include one or more of theaforementioned agents. First reaction mixtures when used duringexamination can be referred to herein as examination reaction mixtures.Optionally, the first reaction mixture comprises a high concentration ofsalt; a high pH; 1, 2, 3, 4, or more types of nucleotides; potassiumglutamate; a chelating agent; a polymerase inhibitor; a catalytic metalion; a non-catalytic metal ion; or any combination thereof. The firstreaction mixture can include 10 mM to 1.6 M of potassium glutamate(including any amount between 10 mM and 1.6 M). Optionally, theincorporation reaction mixture comprises a catalytic metal ion; 1, 2, 3,4, or more types of nucleotides; potassium chloride; a non-catalyticmetal ion; or any combination thereof.

The provided methods can be conducted under reaction conditions thatmodulate the formation and stabilization of a ternary complex during anexamination step. The reaction conditions of the examination steptypically favor the formation and/or stabilization of a ternary complexencapsulating a nucleotide and hinder the formation and/or stabilizationof a binary complex. The binary interaction between the polymerase andtemplate nucleic acid may be manipulated by modulating sequencingreaction parameters such as ionic strength, pH, temperature, or anycombination thereof, or by the addition of a binary complexdestabilizing agent to the reaction. Optionally, high salt (e.g., 50 to1,500 mM) and/or pH changes are utilized to destabilize a binarycomplex. Optionally, a binary complex may form between a polymerase anda template nucleic acid during the examination or incorporation step ofthe sequencing reaction, regardless of the presence of a nucleotide.Optionally, the reaction conditions favor the stabilization of a ternarycomplex and destabilization of a binary complex. By way of example, thepH of the examination reaction mixture can be adjusted from 4.0 to 10.0to favor the stabilization of a ternary complex and destabilization of abinary complex. Optionally, the pH of the examination reaction mixtureis from 4.0 to 6.0. Optionally, the pH of the examination reactionmixture is 6.0 to 10.0.

The provided sequencing methods disclosed herein can function to promotepolymerase interaction with the nucleotides and template nucleic acid ina manner that reveals the identity of the next base while controllingthe chemical addition of a nucleotide. Optionally, the methods areperformed in the absence of detectably labeled nucleotides, or in thepresence of labeled nucleotides wherein the labels are not detected ornot distinguished from each other. Optionally, only the polymeraseharbors a detectable label (e.g., a fluorescent detectable label), andonly the label of the polymerase is detected in the procedure. Again,when the polymerase includes a detectable label, the detectable labelpreferably produces a signal that does not change upon interaction witha cognate or non-cognate nucleotide. For example, the detectable labeldoes not participate in energy transfer to or from a labeled nucleotide,or to or from another label that indicates conformational states of thepolymerase. However, it will be understood that in some embodiments apolymerase having a sequence set forth herein can include a label thatparticipates in energy transfer to or from a labeled nucleotide, or toor from another label that indicates conformational states of thepolymerase.

Provided herein are methods for the formation and/or stabilization of aternary complex comprising a polymerase bound to a primed templatenucleic acid and a nucleotide enclosed within the polymerase-templatenucleic acid complex, under examination reaction conditions. Examinationreaction conditions may inhibit or attenuate nucleotide incorporation.Optionally, incorporation of the enclosed nucleotide is inhibited andthe complex is stabilized or trapped in a pre-chemistry conformation ora ternary complex. Optionally, the enclosed nucleotide is incorporatedand a subsequent nucleotide incorporation is inhibited. In thisinstance, the complex may be stabilized or trapped in apre-translocation conformation. For the sequencing reactions providedherein, the ternary complex is stabilized during the examination step,allowing for controlled nucleotide incorporation. Optionally, astabilized ternary complex is a complex wherein incorporation of anenclosed nucleotide is attenuated, either transiently (e.g., to examinethe complex and then incorporate the nucleotide) or permanently (e.g.,for examination only) during an examination step.

Optionally, the enclosed nucleotide has severely reduced or disabledbinding to the template nucleic acid in the ternary complex. Optionally,the enclosed nucleotide is base-paired to the template nucleic acid at anext base. Optionally, the identity of the polymerase, nucleotide,primer, template nucleic acid, or any combination thereof, affects theinteraction between the enclosed nucleotide and the template nucleicacid in the ternary complex.

Optionally, the enclosed nucleotide is bound to the polymerase of theclosed-complex. Optionally, the enclosed nucleotide is weakly associatedwith the polymerase of the ternary complex. Optionally, the identity ofthe polymerase, nucleotide, primer, template nucleic acid, or anycombination thereof, affects the interaction between the enclosednucleotide and the polymerase in the ternary complex. For a givenpolymerase, each nucleotide may have a different affinity for thepolymerase than another nucleotide. Optionally, a plurality ofnucleotides, for example, all of the nucleotide types that have beenused in reagents of the previous steps of the cycle, is present in awash buffer. Optionally, the plurality of polymerases includes twopolymerases that harbor distinguishable detectable labels, and thepolymerases are components of a combination used with a singlenucleotide. Optionally, this affinity is dependent, in part, on thetemplate nucleic acid and/or the primer.

Optionally, the examination reaction condition comprises a plurality ofprimed template nucleic acids, polymerases, nucleotides, or anycombination thereof. Optionally, the plurality of nucleotides comprisesat least 1, 2, 3, 4, or more types of different nucleotides, for exampledATP, dTTP (or dUTP), dGTP, and dCTP. Alternatively or additionally, theplurality of nucleotides comprises at most 1, 2, 3, or 4 types ofdifferent nucleotides, for example dATP, dTTP (or dUTP), dGTP, and dCTP.Optionally, the plurality of nucleotides comprises one or more types ofnucleotides that, individually or collectively, complement at least 1,2, 3 or 4 types of nucleotides in a template, for example dATP, dTTP (ordUTP), dGTP, or dCTP. Alternatively or additionally, the plurality ofnucleotides comprises one or more types of nucleotides that,individually or collectively, complement at most 1, 2, 3 or 4 types ofnucleotides in a template, for example dATP, dTTP (or dUTP), dGTP, ordCTP. Optionally, the plurality of template nucleic acids is a clonalpopulation of template nucleic acids.

Optionally, the examination reaction mixture comprises one or morereagents or biomolecules generally present in a nucleic acidpolymerization reaction. Reaction components used in addition to thoseset forth herein, may include, but are not limited to, salts, buffers,small molecules, detergents, crowding agents, metals, and ions.Optionally, properties of the reaction mixture may be manipulated, forexample, electrically, magnetically, and/or with vibration.

Useful Nucleotides and Nucleotide Analogs

Optionally, a ternary complex of an examination step comprises either anative nucleotide, or a nucleotide analog or modified nucleotide tofacilitate stabilization of the ternary complex. Optionally, anucleotide analog comprises a nitrogenous base, five-carbon sugar, andphosphate group; wherein any moiety of the nucleotide may be modified,removed and/or replaced. Nucleotide analogs may be non-incorporablenucleotides. Non-incorporable nucleotides may be modified to becomeincorporable at any point during the sequencing method.

Nucleotide analogs include, but are not limited to, alpha-phosphatemodified nucleotides, alpha-beta nucleotide analogs, beta-phosphatemodified nucleotides, beta-gamma nucleotide analogs, gamma-phosphatemodified nucleotides, caged nucleotides, or ddNTPs. Examples ofnucleotide analogs are described in U.S. Pat. No. 8,071,755, which isincorporated by reference herein in its entirety.

Nucleotide analogs can include terminators that reversibly preventnucleotide incorporation at the 3′-end of the primer. One type ofreversible terminator is a 3′-O-blocked reversible terminator. Here theterminator moiety is linked to the oxygen atom of the 3′-OH end of the5-carbon sugar of a nucleotide. For example, U.S. Pat. No. 7,544,794 andU.S. Pat. No. 8,034,923 (the disclosures of these patents areincorporated by reference) describe reversible terminator dNTPs havingthe 3′-OH group replaced by a 3′-ONH₂ group. Another type of reversibleterminator is a 3′-unblocked reversible terminator, wherein theterminator moiety is linked to the nitrogenous base of a nucleotide. Forexample, U.S. Pat. No. 8,808,989 (the disclosure of which isincorporated by reference) discloses particular examples ofbase-modified reversible terminator nucleotides that may be used inconnection with the methods described herein. Other reversibleterminators that similarly can be used in connection with the methodsdescribed herein include those described in U.S. Pat. No. 7,956,171,U.S. Pat. No. 8,071,755, and U.S. Pat. No. 9,399,798 (the disclosures ofthese U.S. patents are incorporated by reference). For reviews ofnucleotide analogs having terminators see e.g., Mu, R., et al., “TheHistory and Advances of Reversible Terminators Used in New Generationsof Sequencing Technology,” Genomics, Proteomics & Bioinformatics11(1):34-40 (2013). Optionally, one or more native nucleotides employedduring the examination step is replaced by a second type of nucleotidethat is incorporated during the incorporation step. For example,nucleotides present in the reaction mixture used during an examinationstep may be replaced by nucleotide analogs that include reversibleterminator moieties (e.g., positioned on the base or sugar of thenucleotide molecule).

Optionally, nucleotide analogs have terminator moieties thatirreversibly prevent nucleotide incorporation at the 3′-end of theprimer. Irreversible nucleotide analogs include2′,3′-dideoxynucleotides, ddNTPs (ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that is essential forpolymerase-mediated synthesis.

Optionally, non-incorporable nucleotides comprise a blocking moiety thatinhibits or prevents the nucleotide from forming a covalent linkage to asecond nucleotide (3′-OH of a primer) during the incorporation step of anucleic acid polymerization reaction. In certain embodiments, theblocking moiety can be removed from the nucleotide, allowing fornucleotide incorporation.

Optionally, a nucleotide analog present in a ternary complex renders theternary complex stable. Optionally, the nucleotide analog isnon-incorporable. Optionally, the nucleotide analog is released and anative nucleotide is incorporated. Optionally, the ternary complex isreleased, the nucleotide analog is modified, and the modified nucleotideanalog is incorporated. Optionally, the ternary complex is releasedunder reaction conditions that modify and/or destabilize the nucleotideanalog in the ternary complex.

Optionally, a nucleotide analog present in a ternary complex isincorporated and the ternary complex is stabilized. The ternary complexmay be stabilized by the nucleotide analog, or for example, by anystabilizing methods disclosed herein. Optionally, the nucleotide analogdoes not allow for the incorporation of a subsequent nucleotide. Theternary complex can be released, for example, by any methods describedherein, and the nucleotide analog is modified. The modified nucleotideanalog may allow for subsequent incorporation of a nucleotide to its3′-end.

Optionally, a nucleotide analog is present in the reaction mixtureduring the examination step. For example, 1, 2, 3, 4 or more nucleotideanalog types are present in the reaction mixture during the examinationstep. Similarly, one or more nucleotide analog types that are present inthe reaction mixture during the examination step can be complementary toat least 1, 2, 3 or 4 nucleotide types in a template nucleic acid.Optionally, a nucleotide analog is replaced, diluted, or sequesteredduring an incorporation step. Optionally, a nucleotide analog isreplaced with a native nucleotide. The native nucleotide may include anext correct nucleotide. Optionally, a nucleotide analog is modifiedduring an incorporation step. The modified nucleotide analog can besimilar to or the same as a native nucleotide.

Any nucleotide modification that traps the polymerase in a ternarycomplex may be used in the methods disclosed herein. The nucleotide maybe trapped permanently or transiently. Optionally, the nucleotide analogis not the means by which a closed-complex is stabilized. Any ternarycomplex stabilization method may be combined in a reaction utilizing anucleotide analog.

Optionally, a nucleotide analog that allows for the stabilization of aclosed-complex is combined with reaction conditions that usually releasethe ternary complex. The conditions include, but are not limited to, thepresence of a release reagent (e.g., catalytic metal ion, such asmagnesium or manganese). Optionally, the ternary complex is stabilizedeven in the presence of a catalytic metal ion. Optionally, the ternarycomplex is released even in the presence of a nucleotide analog.Optionally, the stabilization of the closed-complex is dependent, inpart, on the concentrations and/or identity of the stabilization reagentand/or release reagents, and any combination thereof. Optionally, thestabilization of a ternary complex using nucleotide analogs is combinedwith additional reaction conditions that function to stabilize a ternarycomplex, including, but not limited to, sequestering, removing,reducing, omitting, and/or chelating a catalytic metal ion; the presenceof a polymerase inhibitor, cross-linking agent; and any combinationthereof.

Optionally, one or more nucleotides can be labeled with distinguishingand/or detectable tags or labels. However, in particular embodimentssuch tags or labels preferably are not detected during examination,identification of the base or incorporation of the base, and such tagsor labels are not detected during the sequencing methods disclosedherein. The tags may be distinguishable by means of their differences influorescence, Raman spectrum, charge, mass, refractive index,luminescence, length, or any other measurable property. The tag may beattached to one or more different positions on the nucleotide, so longas the fidelity of binding to the polymerase-nucleic acid complex issufficiently maintained to enable identification of the complementarybase on the template nucleic acid correctly. Optionally, the tag isattached to the nucleobase of the nucleotide. Under suitable reactionconditions, the tagged nucleotides may be enclosed in a ternary complexwith the polymerase and the primed template nucleic acid. Alternatively,a tag is attached to the gamma phosphate position of the nucleotide.

Useful Polymerase Compositions

In certain embodiments, the disclosed approach identifies a cognatenucleotide using the combination of a unique polymerase composition(e.g., a reagent including a polymerase that can be distinguished fromothers, such as a detectably labeled polymerase) and a single nucleotide(e.g., a native nucleotide) without incorporation of the nucleotide.Optionally, a single type of labeled polymerase is used in combinationwith different nucleotides, one at a time, to create the uniquecombinations. Alternatively, more than one distinguishably labeledpolymerase can be used to create the unique polymerase-nucleotidecombinations. While individually labeled polymerases may be used foreach different nucleotide used in an examination step, mixtures of twodifferent labeled polymerases alternatively can be used as a singleunique polymerase composition. Generally speaking, the primer strand ofa primed template nucleic acid molecule undergoing examination ischemically unchanged by the polymerase or any other enzyme duringexamination procedure that identifies the cognate nucleotide. This is tosay that the primer is neither extended by formation of a newphosphodiester bond, nor shortened by nucleolytic degradation during theexamination step to identify the next correct nucleotide.

It is to be understood that four distinguishable polymerase compositionsin accordance with the disclosure do not necessarily require fourdifferent labeled polymerases. For example, two distinguishably labeledpolymerases can be used in combination with two different nucleotides toyield two different polymerase-nucleotide combinations. Alternatively oradditionally, a polymerase having both of the distinguishable labels ora mixture of the same two distinguishably labeled polymerases (i.e.,representing a third distinct polymerase composition) can be used incombination with a third nucleotide to yield a thirdpolymerase-nucleotide combination. Further alternatively oradditionally, an unlabeled polymerase can be used in combination with afourth nucleotide to yield a fourth polymerase-nucleotide combination(i.e., a “dark” combination). In some embodiments, use of a fourthpolymerase-nucleotide combination can be avoided altogether, deducing bythe absence of a signal indicating the cognate nucleotide is any of thefirst three nucleotides that the cognate must be, by default, the fourthnucleotide. By this approach, all four different cognate nucleotides canbe identified using fewer than four different labels. Thus, at most one,two, or three polymerases used in the four polymerase compositions canharbor distinguishable labels. Optionally, four different polymerasesare labeled with four different detectable moieties (e.g., fluorescentmoieties or Raman labels). This approach has successfully allowed forsimultaneous detection of the next correct nucleotide in a multiplexedfield of features by the technique described herein.

Optionally, the polymerase employed during the examination step includesan exogenous detectable label (e.g., a fluorescent label or Ramanscattering tag) chemically linked to the structure of the polymerase bya covalent bond after the polymerase has been at least partiallypurified using protein isolation techniques. For example, the exogenousdetectable label can be chemically linked to the polymerase using a freesulfhydryl or a free amine moiety of the polymerase. This can involvechemical linkage to the polymerase through the side chain of a cysteineresidue, or through the free amino group of the N-terminus. In certainpreferred embodiments, a fluorescent label attached to the polymerase isuseful for locating the polymerase, as may be important for determiningwhether or not the polymerase has localized to a feature or spot on anarray corresponding to immobilized primed template nucleic acid. Thefluorescent signal need not, and preferably does not change absorptionor emission characteristics as the result of binding any nucleotide.Stated differently, the signal emitted by the labeled polymerase ismaintained substantially uniformly in the presence and absence of anynucleotide being investigated as a possible next correct nucleotide.

Optionally, a polymerase in accordance with the present disclosure istagged with a chemiluminescent tag, wherein closed-complex formation ismonitored as a stable luminescence signal in the presence of theappropriate luminescence triggers. The unstable interaction of thepolymerase with the template nucleic acid in the presence of anincorrect nucleotide results in a measurably weaker signal compared tothe ternary complex formed in the presence of the next correctnucleotide. Additionally, an optional wash step prior to triggeringluminescence can remove substantially all polymerase molecules not boundin a stable ternary complex.

Optionally, a polymerase is tagged with an optical scattering tag,wherein ternary complex formation is monitored as a stable opticalscattering signal. The unstable interaction of the polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the ternary complex formed in thepresence of the next correct nucleotide.

Optionally, the polymerase is tagged with a plasmonic nanoparticle tag,wherein the ternary complex formation is monitored as a shift inplasmonic resonance that is different from the plasmonic resonance inthe absence of the ternary complex or the presence of a ternary complexcomprising an incorrect nucleotide. The change in plasmon resonance maybe due to the change in local dielectric environment in the ternarycomplex, or it may be due to the synchronous aggregation of theplasmonic nanoparticles on a cluster of clonally amplified nucleic acidmolecules or another means that affects the plasmons differently in theclosed-complex configuration.

Optionally, the polymerase is tagged with a Raman scattering tag,wherein, the ternary complex formation is monitored as a stable Ramanscattering signal. The unstable interaction of polymerase with thenucleic acid in the presence of an incorrect nucleotide results in ameasurably weaker signal compared to the ternary complex formed in thepresence of the next correct nucleotide.

A common method of introducing a detectable tag on a polymerase involveschemical conjugation to amines or cysteines present in the non-activeregions of the polymerase. Such conjugation methods are well known inthe art. As non-limiting examples, n-hydroxysuccinimide esters (NHSesters) are commonly employed to label amine groups that may be found onan enzyme. Cysteines readily react with thiols or maleimide groups,while carboxyl groups may be reacted with amines by activating them withEDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride).Optionally, N-Hydroxysuccinimide (NHS) chemistry is employed at pHranges where only the N-terminal amines are reactive (for instance, pH7), such that only a single tag is added per polymerase.

Optionally, the tag attached to the polymerase is a charge tag, suchthat the formation of stable ternary complex can be detected byelectrical means by measuring changes in local charge density around thetemplate nucleic acids. Methods for detecting electrical charges arewell known in the art, comprising methods such as field-effecttransistors, dielectric spectroscopy, impedance measurements, and pHmeasurements, among others. Field-effect transistors include, but arenot limited to, ion-sensitive field-effect transistors (ISFET),charge-modulated field-effect transistors, insulated-gate field-effecttransistors, metal oxide semiconductor field-effect transistors andfield-effect transistors fabricated using semiconducting single wallcarbon nanotubes.

Optionally, a charge tag is a peptide tag having an isoelectric pointbelow about 4 or above about 10. Optionally, a polymerase comprising apeptide tag has a total isoelectric point below about 5 or above about9. A charge tag may be any moiety which is positively or negativelycharged. The charge tag may comprise additional moieties including massand/or labels such as dyes. Optionally, the charge tag possesses apositive or negative charge only under certain reaction conditions suchas changes in pH.

A polymerase optionally may be labeled with a fluorophore and/orquencher. Optionally, a nucleic acid is labeled with a fluorophoreand/or quencher. Optionally, one or more nucleotides are labeled with afluorophore and/or quencher. Exemplary fluorophores include, but are notlimited to, fluorescent nanocrystals; quantum dots; green fluorescentprotein and color shifted mutants thereof, phycobiliproteins such asphycocyanin and phycoerythrin, d-Rhodamine acceptor dyes includingdichloro[R110], dichloro[R6G], dichloro[TAMRA], dichloro[ROX] or thelike; fluorescein donor dye including fluorescein, 6-FAM, or the like;Cyanine dyes such as Cy3B; Alexa dyes, SETA dyes, Atto dyes such as alto647N which forms a FRET pair with Cy3B and the like. Fluorophoresinclude, but are not limited to, MDCC(7-diethylamino-3-[([(2-maleimidyl)ethyl]amino)carbonyl]coumarin), TET,HEX, Cy3, TMR, ROX, Texas Red, Cy5, LC red 705 and LC red 640.Fluorophores and methods for their use including attachment topolymerases and other molecules are described in The Molecular Probes®Handbook (Life Technologies, Carlsbad Calif.) and Fluorophores Guide(Promega, Madison, Wis.), which are incorporated herein by reference intheir entireties. Exemplary quenchers include, but are not limited to,ZEN, IBFQ, BHQ-1, BHQ-2, DDQ-I, DDQ-11, Dabcyl, Qxl quencher, Iowa BlackRQ, and IRDye QC-1.

Optionally, binding between a polymerase and a template nucleic acid inthe presence of a correct nucleotide may induce a decrease influorescence, whereas binding with an incorrect nucleotide causes anincrease in fluorescence. Binding between a polymerase and a templatenucleic acid in the presence of a correct nucleotide may induce anincrease in fluorescence, whereas binding with an incorrect nucleotidecauses a decrease in fluorescence. The fluorescent signals may be usedto monitor the kinetics of a nucleotide-induced conformational changeand identify the next base in the template nucleic acid sequence.

Optionally, the polymerase/nucleic-acid interaction may be monitored bya scattering signal originating from the polymerase or tags attached tothe polymerase, for instance, nanoparticle tags.

Use of Polymerase Inhibitors to Stabilize Ternary Complexes

A ternary complex may be formed and/or stabilized by including apolymerase inhibitor in the examination reaction mixture. Inhibitormolecules phosphonoacetate, (phosphonoacetic acid) and phosphonoformate(phosphonoformic acid, common name Foscarnet), Suramin, Aminoglycosides,INDOPY-1 and Tagetitoxin are non-limiting examples of uncompetitive ornoncompetitive inhibitors of polymerase activity. The binding of theinhibitor molecule, near the active site of the enzyme, traps thepolymerase in either a pre-translocation or post-translocation step ofthe nucleotide incorporation cycle, stabilizing the polymerase in itsternary complex conformation before or after the incorporation of anucleotide, and forcing the polymerase to be bound to the templatenucleic acid until the inhibitor molecules are not available in thereaction mixture by removal, dilution or chelation.

Thus, polymerase inhibitor prevents the incorporation of the nucleotidemolecule into the primer of the primer template nucleic acid.Optionally, the inhibitor is a non-competitive inhibitor, an allostericinhibitor, or an uncompetitive allosteric inhibitor. Optionally, thepolymerase inhibitor competes with a catalytic ion binding site in thepolymerase.

Optionally, the polymerase of the ternary complex is prevented fromopening its finger domains and translocating to the next templatenucleic acid position by using pyrophosphate analogs or other relatedmolecules. Pyrophosphate analogs configure the polymerase in ternarycomplex by occupying sites close to the triphosphate binding site in theactive pocket of the polymerase. Release of the pyrophosphate (PPi) iscritical for the polymerase to assume the open conformation, translocateto the next template nucleic acid position, and accept the nextnucleotide. The non-competitive inhibitor, such as Foscarnet(phosphonoformate), phosphonoacetate or other pyrophosphate analogs,traps the polymerase in its fingers-closed conformation. Optionally,binding of the PPi analog is reversible, with the polymerase activityfully restored by washing away, diluting, or sequestering the inhibitorin the reaction mixture. Broadly, any non-competitive inhibitor ofpolymerase activity may be used during the sequencing reaction.

Optionally, a polymerase inhibitor which stabilizes a ternary complex iscombined with reaction conditions which usually release the ternarycomplex, including, but not limited to, the presence of a catalyticmetal ion, such as magnesium or manganese. Optionally, the ternarycomplex is stabilized even in the presence of a catalytic metal ion.Optionally, the ternary complex is released even in the presence of apolymerase inhibitor. Optionally, the stabilization of the ternarycomplex is dependent, in part, on the concentrations, the identity ofthe stabilization reagent, the identity of release reagents, and anycombination thereof. Optionally, the stabilization of a ternary complexusing polymerase inhibitors is combined with additional reactionconditions which also function to stabilize a ternary complex,including, but not limited to, sequestering, removing, reducing,omitting, and/or chelating a catalytic metal ion; the presence of amodified polymerase in the ternary complex; a non-incorporablenucleotide in the ternary complex; and any combination thereof.

Discriminating Conditions: Distinguishing Binary and Ternary ComplexFormation

Optionally, since particular embodiments utilize polymerase bindingwithout incorporation to identify a cognate nucleotide (i.e., the nextcorrect nucleotide), it can be beneficial to enhance discriminationbetween specific- and non-specific polymerase binding to the primedtemplate nucleic acid. This can be achieved, in part, by reducingnon-specific “background” binding due to binary complex formation.

Binary complex formation conveniently can be reduced, inhibited ordestabilized by use of one or more salts that provide monovalentcations. Preferred concentration ranges are from 50 mM to 1,500 mM of asalt that provides monovalent cations (e.g., potassium ions).Preferably, the salt concentration is sufficient to preferentiallydestabilize binary complexes, and to favor ternary complex formationover binary complex formation by at least two-fold, by at leastfive-fold, or even more. Still further, the salt that providesmonovalent cations may further provide a source of dicarboxylate anions,such as glutamate anions. The concentration of the salt that providesthese ions can be from 10 mM to 1.6 M, optionally from 50 mM to 500 mM,or alternatively from 100 mM to 300 mM. Examples of monovalent metalcations include Na⁺ and K⁺; while examples of dicarboxylate anionsinclude glutamate anions (e.g., arising from potassium glutamate).

Stabilizing Ternary Complexes and Controlling Polymerase Exchange

The ability to form and maintain ternary complexes (e.g., produced usingfour different polymerase-nucleotide combinations in serial fashion) ondifferent features of an array can be facilitated by stabilization ofternary complexes. This can be accomplished in a variety of ways.

Optionally, a polymerase is stabilized in its ternary complex by one ora combination of approaches, including: reversible crosslinking of thepolymerase to the nucleic acid; use of allosteric inhibition by smallmolecules, uncompetitive inhibitors, competitive inhibitors, and/ornon-competitive inhibitors; use of non-catalytic cations; use ofaptamers; use of anti-polymerase antibodies; use of a reversibly blockedprimed template nucleic acid molecule (i.e., a non-extendible primer);and denaturation. Optionally, the polymerase inhibitor competes with acatalytic ion binding site in the polymerase. For example,aminoglycosides non-competitively inhibit polymerase activity bydisplacing magnesium binding sites in a Klenow polymerase. Thenon-competitive nature of the interaction with respect to nucleotidebinding allows the polymerase to interact with the template nucleic acidand nucleotide, affecting only the catalytic step of nucleotideincorporation. In all instances, formation of the stabilized ternarycomplex provides information about the identity of the next base on thenucleic acid template. Particularly preferred approaches for trapping orstabilizing the polymerase in a ternary complex include the use ofnon-catalytic cations that inhibit phosphodiester bond formation, suchas non-catalytic lanthanide cations, and/or allosteric inhibitors.

Stabilizing ternary complexes that included primed template nucleicacid, polymerase, and cognate nucleotide is illustrated below by the useof particular non-catalytic metal ions. To determine which non-catalyticmetal cations afforded the longest retention of ternary complexes duringsubsequent binding and wash steps, various candidate cations wereevaluated. Among the metal ions tested in this procedure were: Cu²⁺,Mn²⁺, V⁵⁺, Eu³⁺, Ni²⁺, Sr²⁺, Tb³⁺, Ca²⁺ and Co²⁺. Certain preferredreaction conditions substantially maintain ternary complex signals inthe absence of non-bound polymerase (i.e., polymerase free in solution,not bound to any immobilized template) over an extended period (e.g., ofgreater than about 30 seconds, such as about 30-60 seconds). Forexample, ternary complex binding signal measured at the desired timepoint following a wash step can be expressed as a percentage of themaximum signal (using the signal measured at the time of initialnucleotide contact as a baseline). Preferred metal ions includetrivalent lanthanide ions, including europium ions and terbium ions.Results confirmed that superior retention of ternary complexes on primedtemplate nucleic acid molecules by these cations were attributable tothe physiochemical properties of trivalent lanthanides.

A blocked primer terminating at its 3′-end with a reversible terminatornucleotide that precludes phosphodiester bond formation also can be usedfor stabilizing ternary complex formation. Indeed, the product of areaction that incorporates either a reversible or irreversibleterminator nucleotide includes blocked primers that stabilize ternarycomplexes. In any reaction step described above, formation of astabilized ternary complex containing a nucleotide that is notincorporated may be monitored to identify the next correct base in thenucleic acid sequence. Reaction conditions can be changed to disengagethe polymerase and cognate nucleotide from a blocked primed templatenucleic acid molecule, and changed again to remove from the localenvironment any reversible terminator moiety attached to the nucleotideat the 3′-end of the primer strand of the primed template nucleic acidmolecule. In some embodiments, both the polymerase and cognatenucleotide of the ternary complex, and the reversible terminator moietyare removed in a single step using a reagent that dissociates ternarycomplexes and cleaves the reversible terminator moiety from its positionat the 3′-end of the blocked primed template nucleic acid molecule.

Systems

The disclosed technique for determining cognate nucleotides usingengineered polymerases, whether for a single nucleic acid feature or fora population of different nucleic acid features spaced apart in a flowcell or well of a multiwell plate, can be performed using a dedicatedsystem of interrelated modules or components. Some useful systems willbe familiar to those having an ordinary level of skill in the art, andcan be adapted or configured for processing by the disclosed techniquethat relies on identification or tracking of distinguishably labeledpolymerases. An exemplary system for use in identifying a next correctnucleotide of a primed template nucleic acid molecule typically willinclude: a reaction vessel; a reagent dispense module; an imagingmodule; a processing module; and an electronic storage device. Systemsuseful for single-scan imaging of a population of nucleic acid featureswill have the capability of detecting four different fluorescentemission wavelengths. Essential features of particularly preferredsystems are described below.

The reaction vessel employed in the system may take different forms. Thereaction vessel will be in fluid communication with a supply of one ormore labeled polymerases. Examples of reaction vessels include flowcells having inlet and outlet ports, and one or more wells of amultiwell plate. Contained within the reaction vessel will be acollection or population of nucleic acid features to be processed by thedisclosed technique. The nucleic acid features may be “clusters” ofspaced-apart amplified nucleic acids (e.g., in situ amplified nucleicacids). Alternatively, individual beads harboring homogenous populationsof nucleic acids may be contained within the reaction vessels.

The reagent dispense module also may take different forms. The reagentdispense module directs into the reaction vessel, one at a time, aliquid reagent that includes one of the labeled polymerases incombination with one or more different nucleotides for each of aplurality of reagent exchanges. Optionally, the labeled polymerases aredistinguishably labeled polymerases that harbor different fluorescentdetectable labels. Optionally, none of the fluorescent detectable labelsis an intercalating dye, and none of the fluorescent detectable labelsis excited by energy transfer from a different molecular species.Optionally, the reaction vessel is a flow cell, and each reagentexchange involves flowing through the flow cell a second liquid reagentto replace a first liquid reagent. Optionally, the reagent dispensemodule includes a syringe pump that controllably transfers one of thefour distinguishably labeled polymerases in combination with one or moreof four different nucleotides. Optionally, the liquid reagent directedinto the reaction vessel by the reagent dispense module includes aternary complex-stabilizing agent. Exemplary ternary complex-stabilizingagents are disclosed elsewhere, herein.

The imaging module also may take different forms. The imaging modulewill be capable of detecting which of the four distinguishably labeledpolymerases is present in a complex that includes: (i) the primedtemplate nucleic acid molecule; (ii) one of the four distinguishablylabeled polymerases; and (iii) the next correct nucleotide. Optionally,the imaging module includes an illumination component and a detectioncomponent. Illumination components may take the form of light emittingdiodes (LEDs) that generate a range of wavelengths. A plurality ofdifferent LEDs may be employed in the imaging module. Useful detectorsinclude fluorometers that measure parameters of fluorescence. There alsocan be one or more optical filters for narrowing the range or band ofwavelengths that are transmitted either to a sample or to a detector.The detection component of the imaging module optionally can beconfigured to detect intensities of a plurality of differentwavelengths, each corresponding to a fluorescence emission by one of thefour distinguishably labeled polymerases. Thus, each of the fluorescentdetectable labels associated with one of the polymerases can be excitedby a wavelength of energy produced by the illumination component (e.g.,produced by one of the LEDs), and an emission signal produced by thedetectable label can be detected by the detection component. In oneembodiment, the imaging module includes an illumination component and adetection component, where each of four distinguishably labeledpolymerases is labeled with a fluorescent detectable label, where eachof the fluorescent detectable labels is excited by a wavelength ofenergy produced by the illumination component, and where the detectioncomponent is configured to detect intensities of a plurality ofdifferent wavelengths, each corresponding to a fluorescence emission byone of the four distinguishably labeled polymerases.

The processing module also can take different forms. For example, theprocessing module can include a computer (e.g., either a standalonecomputer or processor, a computer or processor integrated into thesystem within a common housing or chassis) configured with software tocompare intensities of the plurality of different wavelengths, and todetermine therefrom the identity of the next correct nucleotide. Theprocessing module will be configured to receive a result from theimaging module, and further configured to identify the next correctnucleotide using the result processed result. Configuring of theprocessing module may involve embedded, or otherwise accessible softwareinstructions (e.g., being accessed from a remote software repository).

The electronic storage device also can take different forms. The storagedevice will be in communication with the processing module, and canstore a non-transient record of the next correct nucleotide identifiedby the processing module. For example, the electronic storage device canbe a computer hard drive, flash drive, floppy disk, compact disk (CD) orother optical disk storage medium, cloud storage arrangement, and thelike.

Optionally, the system can also include an output device that produces anon-transient record of the next correct nucleotide identified by theprocessing module. The non-transient record produced by the outputdevice optionally can be either a record stored on computer-readablemedia, or a record printed on paper.

EXAMPLES

Following are illustrations showing how polymerases in accordance withthe disclosure can be used in procedures for identifying one or morecognate nucleotides in the sequence of a primed template nucleic acid.Notably, engineered polymerases for each of the named mutant categoriesin Table 1 were prepared and tested for interaction with primed templatenucleic acid in the presence of cognate or non-cognate nucleotides, andfor the ability to catalyze phosphodiester bond formation (i.e.,incorporate nucleotide into the primed template nucleic acid). In allcases, testing was conducted using polymerases that included theextraneous N-terminal stretch of amino acids represented by SEQ ID NO:6.Since this portion of the engineered polymerase does not participate innucleotide binding or interaction with the primed template nucleic acid,inclusion of the sequence of SEQ ID NO:6, or portions thereof, byattachment to variants of the sequence of SEQ ID NO:3 is optional.

Example 1 describes the use of a TQE polymerase in Sequencing ByBinding™ protocols involving cycles of examination to identify cognatenucleotides. Results demonstrated that the engineered enzyme exhibitedsubstantially reduced non-specific DNA binding in the absence of cognatenucleotide while retaining the ability to incorporate cognate nucleotidein the presence of the catalytic Mg²⁺ metal ion. As described above, theTQE mutant used in the demonstration included a single amino acid changerelative to the polypeptide sequence of the related CBT parent enzymehaving the polypeptide sequence of SEQ ID NO:1. Similarly, the CBTparent polymerase having the polypeptide sequence of SEQ ID NO:1 alsowas prepared and purified.

Example 1 Demonstration of Cognate Nucleotide Identification with LowNon-Specific DNA Binding Polymerase

The above-described TQE mutant polymerase was prepared and purifiedusing standard techniques that will be familiar to those having anordinary level of skill in the art. The purified TQE mutant polymerasehad the sequence of SEQ ID NO:1, except that that the amino acid atposition 307 was Glu (E) instead of Gln (Q). None of the proteinsequence modifications upstream of the first methionine of the Bst-f DNApolymerase (i.e., position 27 of SEQ ID NO:1; or SEQ ID NO:3) was deemedessential for the desired combination of reduced non-specific DNAbinding in the absence of cognate nucleotide and for Mg²⁺-catalyzedincorporation in the presence of cognate nucleotide. Thus, inclusion ofthese modifications is optional in the working product.

A FORTEBIO® (Menlo Park, Calif.) Octet instrument employing biolayerinterferometry to measure binding reactions at the surface of a fiberoptic tip was used in a multiwell plate format to illustrate propertiesof the polymerase in the context of a nucleic acid sequencing technique.Primed template nucleic acid molecules biotinylated at the 5′-ends ofthe template strand were immobilized onto fiber optic tipsfunctionalized with streptavidin (SA) using standard procedures. Theprimed template nucleic acid molecule in this procedure had TA as thenext two correct nucleotides downstream of the primer.

The cycling procedure involved steps for: (1) washing/regeneratingsensor tips; (2) contacting the template with one of four native dNTPsto investigate complex formation; (3) washing with an EDTA solution tostrip complexes from the primed template nucleic acid molecule. Anincorporation step followed a complete round of binding and examinationusing the four dNTPs, one at a time. Sensor tips were washed/regeneratedin a Tris-buffered solution (pH 8.0) that included KCl, potassiumglutamate, and 0.01% Tween-20 before commencing the cycling protocol.The first incoming nucleotide was interrogated with 500 nM of either TQEor 500 nM of CBT in the presence of examination buffer (30 mM Tris-HCl(pH 8.0); either 50, 100 or 150 mM KCl; 320 mM potassium glutamate; 2 mMSrCl₂; 0.01% Tween-20; 0.1 mg/mL acetylated BSA; and 1 mMβ-mercaptoethanol). Native nucleotides were employed in the procedure,and were contacted to the sensor tip in the following order: dATP, dTTP,dGTP, and dCTP. Each of the dNTPs was present at a concentration of 100μM, except for dTTP, which was used at a concentration of 200 μM.Nucleotide binding steps were for a period of about 30 seconds at 30° C.At the end of each nucleotide binding and examination step, any formedcomplexes were washed from the sensor tip for 45 seconds using an EDTAsolution containing KCl to chelate divalent cations. Thereafter, thebiosensor was regenerated for 30 seconds before moving to the next dNTPexam.

Following examination of all four dNTPs to determine whether a ternarycomplex had formed, incorporation reactions were performed to comparepolymerase activity of the TQE mutant with the CBT parent enzyme. First,ternary complexes were prepared by contacting the sensor tips with thecognate nucleotide (i.e., dTTP) at a concentration of 200 μM for 30seconds. Next, biosensor tips were transferred to an incorporationbuffer (30 mM Tris-HCl (pH 8.0), 50 mM KCl, 50 mM Mg²⁺) for 30 seconds.Finally, complexes were washed from the sensor tips for 45 seconds usingthe EDTA solution containing KCl to chelate divalent cations. Again, thebiosensor was regenerated for 30 seconds before moving to the nextseries of examination reactions using all four dNTPs, one at a time.Results from this latter set of examination reactions was informativeregarding binding and incorporation activities of the mutant enzyme.

Results from the procedure, shown in FIGS. 1A-1C, confirmed that thespecificity-enhanced TQE polymerase correctly identified the cognatenucleotide, bound the DNA template with substantially reduced affinityin the absence of cognate nucleotide, and correctly incorporated cognatenucleotide in the presence of the catalytic Mg²⁺ metal ion. The figuresshow examination traces for all four nucleotides conducted using the TQEand CBT polymerases under three different buffer conditions. Ternarycomplexes generated in the presence of dTTP indicated that bothpolymerases correctly identified the cognate nucleotide. In all cases,non-cognate nucleotides were associated with substantially reducedbinding signals for the TQE enzyme compared with the CBT parent.Following the step to permit incorporation, both the CBT and TQE enzymeswere shown to possess catalytic activity. In both cases, the subsequentnucleotide (dATP) was properly identified. This indicated that cognatenucleotide had been incorporated efficiently by the mutant enzyme underincorporating conditions. Of course, a repetitive cycling procedure toconduct extensive sequence determination can use a different enzyme forthe incorporation step. A reversible terminator nucleotide (e.g., anunlabeled reversible terminator nucleotide) may be used in theincorporation procedure. Optionally, different polymerase enzymes can beused to incorporate reversible terminator nucleotides and perform theexamination steps.

Example 2 describes use of the UQE specificity-enhanced polymerase inSequencing By Binding protocols involving cycles of examination toidentify cognate nucleotides. Results demonstrated that the engineeredUQE enzyme exhibited substantially reduced non-specific DNA binding inthe absence of cognate nucleotide while retaining the ability toincorporate cognate nucleotide in the presence of the catalytic Mg²⁺metal ion. As described above, the UQE mutant includes a single aminoacid change at position 314 of the modified CBU enzyme identified by SEQID NO:13. Again, this CBU parent enzyme included an exogenous cysteineresidue and N-terminal His-tag.

Example 2 Demonstration of Cognate Nucleotide Identification with LowNon-Specific DNA Binding Polymerase

The UQE mutant polymerase having the polypeptide sequence of SEQ IDNO:13, except for replacement of Gln (Q) by Glu (E) at position 314, wasprepared and purified using standard molecular cloning, gene expression,and protein purification techniques that will be familiar to thosehaving an ordinary level of skill in the art. Similarly, the CBU parentpolymerase having the polypeptide sequence of SEQ ID NO:13 also wasprepared and purified.

The procedures of Example 1 were followed, substituting the UQEpolymerase in place of the TQE polymerase, and substituting the CBUpolymerase in place of the CBT polymerase.

Results from the procedure, shown in FIG. 2, confirmed that thespecificity-enhanced polymerase correctly identified the cognatenucleotide, bound the DNA template with substantially reduced binding inthe absence of cognate nucleotide, and correctly incorporated cognatenucleotide in the presence of the catalytic Mg²⁺ metal ion. The figureshows examination traces for all four nucleotides conducted using theUQE and CBU polymerases under three different buffer conditions. Ternarycomplexes generated in the presence of dTTP indicated that bothpolymerases correctly identified the cognate nucleotide. In all cases,non-cognate nucleotides were associated with substantially reducedbinding signals for the UQE enzyme compared with the CBU parent.Following the step to permit incorporation, both the CBU and UQE enzymeswere shown to possess catalytic activity. In both cases, the subsequentnucleotide (dATP) was properly identified. This indicated that cognatenucleotide had been incorporated efficiently by the mutant enzyme underincorporating conditions. Of course, a repetitive cycling procedure toconduct extensive sequence determination can use a different enzyme forthe incorporation step. A reversible terminator nucleotide (e.g., anunlabeled reversible terminator nucleotide) may be used in theincorporation procedure. Optionally, different polymerase enzymes can beused to incorporate reversible terminator nucleotides and perform theexamination steps.

The foregoing discussion of DNA polymerase mutants addressed instanceswherein as few as a single amino acid change could distinguished aspecificity-enhanced polymerase (e.g., a low background DNA bindingpolymerase) from its parent enzyme. Surprisingly, amino acid changesintroduced into these mutants were in a region of the enzyme notpreviously known to exhibit sequence conservation suggestive offunctional importance.

Following is a description of another mutant DNA polymerase, where thispolymerase contained two amino acid changes relative to the parentpolymerase. More particularly, the DSA mutant polymerase possessesincreased nucleotide discrimination between correct and incorrectnucleotides. The DSA mutant was made by site-directed mutagenesis of thepolynucleotide encoding the CBT polymerase of SEQ ID NO:1 so that aminoacid positions 276 and 451 were both occupied by Cys residues. Theseregions of the polymerase altered by these changes are believed to be atthe tip of the thumb and finger domains of the Bst-f polymerase. Theconsequence of the two altered positions was decreased binary backgroundbinding.

Example 3 describes use of the DSA specificity-enhanced polymerase inSequencing By Binding protocols involving cycles of examination toidentify cognate nucleotides. Results demonstrated that the engineeredDSA enzyme exhibited substantially reduced non-specific DNA binding inthe absence of cognate nucleotide while retaining the ability toincorporate cognate nucleotide in the presence of the catalytic Mg²⁺metal ion. As demonstrated below, the DSA polymerase advantageously gavea disproportionately large decrease in its binary and incorrect ternarybinding compared to its correct ternary binding. Therefore, the DSAenzyme was capable of increased discrimination of ternary complexeshaving cognate nucleotides by lowering background binding that was dueto binary complex formation. The DSA mutant includes two amino acidchanges at positions 276 and 451 of the modified CBU enzyme identifiedby SEQ ID NO:1. Again, this CBU parent enzyme included an exogenouscysteine residue and N-terminal His-tag.

Example 3 Demonstration of Cognate Nucleotide Identification with LowNon-Specific DNA Binding Polymerase

The DSA mutant polymerase having the polypeptide sequence of SEQ IDNO:1, except for replacement of Lys (K) and Gln (Q) by Cys (C) at eachof positions 276 and 451, was prepared and purified using standardmolecular cloning, gene expression, and protein purification techniquesthat will be familiar to those having an ordinary level of skill in theart. Similarly, the CBT parent polymerase having the polypeptidesequence of SEQ ID NO:1 also was prepared and purified.

A modification of the procedure in Example 1 was followed to assesspolymerase activity, substituting the DSA polymerase in place of the TQEpolymerase and adding a single step. The cycling procedure involvedsteps for: (1) washing/regenerating sensor tips; (2) contacting thetemplate with a solution containing polymerase but no nucleotide; (3)contacting the template with one of four native dNTPs to investigatecomplex formation; and (4) washing with an EDTA solution to stripcomplexes from the primed template nucleic acid molecule. Anincorporation step followed a complete round of binding and examinationusing the four dNTPs, one at a time. Sensor tips were washed/regeneratedin a Tris-buffered solution (pH 8.0) that included KCl, potassiumglutamate, 1 mM SrCl₂, 0.01% Tween-20 before commencing the cyclingprotocol. Binary complex formation was permitted by contacting sensortips with wash/regeneration solution containing either 500 nM of DSApolymerase or 500 nM of CBT polymerase, but not containing anynucleotide. The first incoming nucleotide was interrogated with 500 nMof either DSA or 500 nM of CBT in the presence of examination buffer (30mM Tris-HCl (pH 8.0); either 100, 200 or 400 mM KCl; 320 mM potassiumglutamate; 2 mM SrCl₂; 0.01% Tween-20; 0.1 mg/mL acetylated BSA; and 1mM β-mercaptoethanol). Native nucleotides were employed in theprocedure, and were contacted to the sensor tip in the following order:dATP, dTTP, dGTP, and dCTP. Each of the dNTPs was present at aconcentration of 100 μM, except for dTTP, which was used at aconcentration of 200 μM. Nucleotide binding steps were for a period ofabout 15 seconds at 30° C. At the end of each nucleotide binding andexamination step, any formed complexes were washed from the sensor tipfor 45 seconds using a solution containing EDTA to chelate divalentcations. Thereafter, the biosensor was regenerated for 30 seconds beforemoving to the next dNTP exam.

Following examination of all four dNTPs to determine whether a ternarycomplex had formed, incorporation reactions were performed to comparepolymerase activity of the DSA mutant with the CBT parent enzyme. First,ternary complexes were prepared by contacting the sensor tips with thecognate nucleotide (i.e., dATP) at a concentration of 100 μM for 30seconds. Next, biosensor tips were transferred to an incorporationbuffer (30 mM Tris-HCl (pH 8.0), 50 mM KCl, 50 mM MgCl₂) for 15 seconds.Finally, complexes were washed from the sensor tips for 45 seconds usingthe EDTA solution to chelate divalent cations. Again, the biosensor wasregenerated before moving to the next series of examination reactionsusing all four dNTPs, one at a time. Results from this latter set ofexamination reactions was informative regarding binding andincorporation activities of the mutant enzyme.

Results from the procedure, shown in FIG. 3, confirmed that thespecificity-enhanced polymerase correctly identified the cognatenucleotide, bound the DNA template with substantially reduced binding inthe absence of cognate nucleotide, and correctly incorporated cognatenucleotide in the presence of the catalytic Mg²⁺ metal ion. The figureshows examination traces for all four nucleotides conducted using theDSA and CBT polymerases under three different buffer conditions. Ternarycomplexes generated in the presence of dATP indicated that bothpolymerases correctly identified the cognate nucleotide. Notably, themagnitude of the signal for DSA binding to DNA in the absence orpresence of cognate nucleotide was generally lower than the signal forCBT binding to DNA (i.e., for both binary and ternary complexformation). DSA binding showed a disproportionally larger decrease inits binary signal compared to signal resulting from ternary complexformation. Therefore, despite the overall lower signal, the enzyme wascapable of increased discrimination by lowering background binding.Surprisingly, the DSA polymerase also gave better discrimination betweenbinary and ternary complex formation at lower concentrations of saltthan CBT. Still further, results from the incorporation step confirmedthat both the DSA and CBT enzymes possessed catalytic activity. In bothcases, the subsequent nucleotide (dTTP) was properly identified. Thisindicated that cognate nucleotide had been incorporated efficiently bythe mutant enzyme under incorporating conditions. Of course, arepetitive cycling procedure to conduct extensive sequence determinationcan use a different enzyme for the incorporation step. A reversibleterminator nucleotide (e.g., an unlabeled reversible terminatornucleotide) may be used in the incorporation procedure. Optionally,different polymerase enzymes can be used to incorporate reversibleterminator nucleotides and perform the examination steps.

Example 4 describes procedures illustrating the use of detectablylabeled polymerases for determining cognate nucleotide identity. TheSequencing By Binding™ protocol in this Example employed label-freenative nucleotides, and label-free primed template nucleic acids. Thesequencing protocol was carried out by flowing different reagentsthrough a flow cell containing immobilized primed template nucleicacids. Although individual types of nucleotides (i.e., either dATP,dGTP, dCTP, or dTTP) were tested one at a time for ternary complexformation with the primed template nucleic acid and labeled polymerase,an alternative protocol employs simultaneous testing of two or moredistinguishably labeled polymerases (e.g., CBT, TQE, or DSApolymerases). Engineered polymerases in this Example were constructedusing the scaffold of SEQ ID NO:1, including the amino acidsubstitutions indicated in Table 1, to permit convenient proteinpurification and fluorescent labeling. The same labeling could have beencarried out using the thrombin cleavage product scaffold of SEQ ID NO:2,and is compatible with this procedure.

Example 4 Nucleic Acid Sequence Determination Using EngineeredPolymerases Having Fluorescent Labels

Nucleic acid features used as templates in a nucleic acid sequencingapplication were synthesized in situ within a flow cell using a rollingcircle amplification (RCA) protocol. Immobilized primers hybridized tosingle-stranded circular templates were used to generate strands ofsequencing templates. Immobilized strands were hybridized tocomplementary sequencing primers and then used in a Sequencing ByBinding™ procedure. Sequencing primers were blocked from extension attheir 3′-ends by incorporating reversible terminator nucleotides having3′ aminooxy (—ONH₂) blocking groups. A single type of template yieldingTAGCATCAGA (SEQ ID NO:7) as the sequence to be determined was used inprocedures with the CBT polymerase. Two different templates yieldingCCCTGTCATG (SEQ ID NO:8) and CCCATTTATG (SEQ ID NO:9) as the sequencesto be determined were used in procedures with the TQE polymerase.Similarly, two different templates yielding CCGATTCGTC (SEQ ID NO:10)and CCATGTTTCA (SEQ ID NO:11) as the sequences to be determined wereused in procedures with the DSA polymerase.

A reagent cycling procedure with continuous fluorescence monitoring wasused for assessing cognate nucleotide identification. Solutionscontaining a single type of nucleotide (dATP, dGTP, dCTP, or dTTP) incombination with either fluorescently labeled CBT polymerase,fluorescently labeled TQE polymerase, or fluorescently labeled DSApolymerase were flowed into the flow cell one at a time to permitformation and detection of ternary complexes. Polymerases were labeledusing standard maleimide chemistry for covalent attachment of a Cy-5moiety to the thiol functional group of an engineered Cys residue nearthe N-terminus. Flows of nucleotides were ordered as: dATP, dGTP, dCTP,and dTTP. All solutions used for these examination steps included Trisbuffer (pH 8.0), KCl, trehalose, 1,2-propanediol, hydroxylamine, DMSO,Sr²⁺ ion, F-127 detergent, 100 μM label-free dNTP (i.e., native dNTP),and 20 nM polymerase (i.e., either CBT, TQE, or DSA). Examinationsolutions containing the CBT polymerase were adjusted to include 240 mMKCl and 80 mM potassium glutamate; solutions containing the TQEpolymerase were adjusted to include 180 mM KCl, and no potassiumglutamate; while solutions containing the DSA polymerase were adjustedto include 50 mM KCl, and no potassium glutamate. Following eachexamination step to detect fluorescence associated with nucleic acidfeatures during one of the nucleotide and polymerase flows, the flowcell was washed with a regeneration buffer that included Tris buffer (pH8.0), 50 mM KCl, trehalose, 1,2-propanediol, hydroxylamine, DMSO, Sr²⁺ion, and F-127 detergent. This was followed by a wash with a quenchingsolution that included Tris buffer (pH 8.0), NaCl, Tween-20, SDS, 2 mMeach of EDTA and NTA metal ion chelators, and hydroxylamine. Thisprocess was cycled four times to permit interrogation of each differentnucleotide. Following each set of four examination reactions, 3′-ONH₂blocking groups were removed from the primers using an acetate-buffered(pH 5.5) cleavage reagent that included NaNO₂ and TCEP. The nextreversible terminator nucleotide was incorporated using a pH-bufferedreaction mixture that included all four label-free reversible terminatornucleotides (i.e., dNTP-ONH₂) in a solution that included TherminatorDNA polymerase (New England Biolabs; Ipswich, Mass.) and MgCl₂. Allprocedures were carried out at 47° C. Signals arising from fluorescentpolymerase associating with immobilized nucleic acid features in thepresence of different nucleotides were monitored and recorded throughoutthe procedure using a fluorescent microscope configured with a digitalcamera that detected emission from the Cy-5 fluorescent moiety joined tothe polymerase. Pixels measured from captured images as a function oftime were plotted to determine cognate nucleotide identity. In oneapproach, the nucleotide giving the highest magnitude fluorescent signalwas identified as the cognate nucleotide.

Results from this procedure showed that the TQE and DSA polymerasesadvantageously discriminated between cognate and non-cognate nucleotidesunder lower salt conditions compared to the CBT polymerase. Thisresulted in higher signal intensities. Procedures carried out using theparent CBT polymerase (see FIG. 4A) gave evidence for “read-ahead,”where signals were detected for both the next correct base (i.e., “n+1”)as well as for the subsequent base (i.e., “n+2”). For example, thehighest signal among the first set of four nucleotides tested wasassociated with dTTP (the n+1 position), and the second highest signalwas associated with dATP (the n+2 position). This feature of the CBTpolymerase was substantially less apparent in results obtained using theTQE and DSA polymerases (see FIGS. 4B-4C). Signal-to-backgroundmeasurements were generally higher when using the DSA polymerase, and soadvantageously favored correct nucleotide identification using basecalling algorithms where maximal peak height identified cognatenucleotide. Use of the DSA polymerase was also associated with signalsof more uniform magnitude over more extended read lengths when comparedwith the CBT polymerase. Significantly, compared with results obtainedusing the CBT polymerase, and even the TQE polymerase, the DSApolymerase discriminated between cognate and non-cognate nucleotidesunder substantially lower salt conditions.

It was discovered during development of the presently disclosedtechniques that there are advantages to achieving discrimination betweenformation of binary and ternary complexes under low salt conditions(e.g., where the concentration of salt providing monovalent cations isin the range of from 10 mM to 500 mM, or even from 10 mM to 250 mM. Forexample, the higher salt conditions frequently are used to achieve gooddiscrimination between binary and ternary complex formation can lead tocompaction of the sequencing template, thereby restricting polymeraseaccess undesirably. Accordingly, certain preferred polymerases exhibitedenhanced discrimination between cognate and non-cognate nucleotidebinding under conditions where the concentration of KCl is below 250 mMwhen the concentration of potassium glutamate is below 350 mM.

Results presented herein demonstrated the benefits of performingSequencing By Binding™ procedures using engineered polymerasescharacterized by low background DNA binding. Example polymerases havingthese features included the TQE and DSA polymerases, whichadvantageously retained the ability to incorporate cognate nucleotide.As indicated above, the DSA polymerase was further characterized by anability to operate under low salt conditions that facilitated longerread lengths, possibly due to effects on the sequencing template.Significantly, the nature of the mutations characterizing these twomutant polymerases suggested that the mechanisms underlying changedfunctional activities relative to the parent polymerases were different.Different mutations resulting in novel characteristics were nextcombined into a single polymerase with the intention of achieving asynergistic effect that might not be possible when different mutationsaffected the same polymerase functionality.

Example 5 describes procedures showing how independentbackground-reducing mutations were combined in a single engineeredpolymerase that was used in Sequencing By Binding™ protocols. Thisengineered polymerase is referred to herein as “TEE.”

Example 5 Polymerase Engineered for Enhanced Discrimination inSequencing by Binding™ Protocols

Conventional laboratory techniques that will be familiar to those havingan ordinary level of skill in the art of molecular biology and proteinpurification were used to produce the TEE polymerase, which includedmutated positions 250 (K to C), 281 (Q to E), and 425 (Q to C) in thescaffold of SEQ ID NO:3. Since the sequence of SEQ ID NO:3 is fullycontained within the sequences of each of SEQ ID Nos:1-2, the amino acidreplacements corresponded to positions 259, 290 and 434 of SEQ ID NO:2;and to positions 276, 307 and 451 of SEQ ID NO:1. Again, the scaffold ofSEQ ID NO:1 included an N-terminal polypeptide sequence that aided inprotein purification, and SEQ ID NO:2 represented the thrombin cleavageproduct of SEQ ID NO:1. To illustrate flexibility in the nature ofpolymerases that can be used, the polypeptide that included theextraneous polyhistidine motif was used to demonstrate functionalsimilarities and differences with respect to the parent DSA polymerase.

Activity of the engineered TEE polymerase was investigated using thebiolayer interferometry technique, essentially as described herein underExample 1. The first two cognate nucleotides for the sensor-immobilizedtemplate undergoing testing were dATP followed by dTTP. After initialloading of the primed template nucleic acid molecule, and washing toremove material that did not immobilize, the optical sensor tip wascycled through exposure to various reagents to permit assessment ofbinary and ternary complex formation. The cycles included exposure tothe TEE polymerase in the absence of nucleotide (to permit formation ofa binary complex); exposure to the combination of the TEE polymerase andan unlabeled test dNTP (to permit formation of a ternary complex whenthe test dNTP is the next correct nucleotide); stripping of allcomplexes from the sensor tip using an EDTA solution; and regeneratingthe tip with a washing/regenerating solution to remove traces of EDTA.In this instance one nucleotide at a time was used, with the order ofexposure being: dATP, dTTP, dGTP, and dCTP. Alternative procedures canemploy nucleotide combinations (e.g., pairwise combinations of differentnucleotides). Examination conditions used in this procedure included 30mM Tris-HCl (pH 8.0); 100 mM KCl; 320 mM potassium glutamate; 2 mMSrCl₂; 0.01% Tween-20; 0.1 mg/mL acetylated BSA; 1 mM β-mercaptoethanol;and 900 nM TEE polymerase.

Comparative results were obtained using the DSA polymerase in place ofthe TEE polymerase, where the procedures were conducted in parallel. TheDSA polymerase used in the procedure included the extraneouspolyhistidine-tag motif that also was present in the TEE polymerase usedin the procedure. This meant that the two polymerases differed at only asingle amino acid position. The DSA trial was carried out using 300 nMDSA in place of TEE. Signal magnitudes (peak heights) for cognatenucleotides were compared to the highest signal magnitude measured forincorrect nucleotides.

Following the first round of examination for all four nucleotides, anincorporation reaction was performed using each of the polymerases andonly the next correct nucleotide, dATP. A single nucleotide incorporatedinto the primed template nucleic acid because the following cognatenucleotide for the synthetic template used in the procedure was dTTP.Procedures used for the incorporation reaction were essentially asdescribed under Example 1. After the incorporation was complete, cyclesof washing to regenerate the sensor tip; exposure to either the TEE orDSA polymerase in the absence of nucleotide to permit binary complexformation; and exposure to a test dNTP to investigate ternary complexformation were resumed.

Results from the procedure, shown in FIG. 5, demonstrated that theengineered TEE polymerase advantageously exhibited very low signalindicating binary complex formation, and high signal indicating ternarycomplex formation. The TEE polymerase also retained the ability toincorporate cognate nucleotide efficiently. As indicated in FIG. 5, thepost-incorporation nucleotide-binding cycles clearly indicated that thenext correct nucleotide was dTTP. This could only have resultedfollowing incorporation of the preceding nucleotide (i.e., dATP). Theratio of correct-to-highest incorrect signal during the first round ofexamination conducted using four nucleotides was 2.75 for TEE (i.e.,1.10 vs. 0.4), and 1.95 for DSA (i.e., 1.75 vs. 0.9). The ratio ofcorrect-to-highest incorrect signal during the second round ofexamination conducted using four nucleotides was 4.83 for TEE (i.e.,1.45 vs. 0.3), and 1.60 for DSA (i.e., 1.00 vs. 0.6). These latterresults indicated that the engineered TEE polymerase exhibited improveddiscrimination between cognate and non-cognate nucleotides relative tothe DSA parent polymerase. Improved discrimination can be an advantage,for example when using the polymerase in Sequencing By Binding™ protocolthat identifies cognate nucleotide without incorporation.

This invention has been described with reference to a number of specificexamples and embodiments thereof. Of course, a number of differentembodiments of the present invention will suggest themselves to thosehaving ordinary skill in the art upon review of the foregoing detaileddescription. Thus, the true scope of the present invention is to bedetermined upon reference to the appended claims.

What is claimed is:
 1. An engineered DNA polymerase, comprising avariant of the sequence of SEQ ID NO:3, said variant being at least 80%identical to SEQ ID NO:3 and comprising an amino acid substitutionmutation at one or more of positions K250, Q281, D355, Q425, and D532.2. The engineered DNA polymerase of claim 1, wherein the variant is atleast 90% identical to SEQ ID NO:3.
 3. The engineered DNA polymerase ofclaim 2, wherein the variant is at least 95% identical to SEQ ID NO:3.4. The engineered DNA polymerase of claim 3, wherein the variant is atleast 98% identical to SEQ ID NO:3.
 5. The engineered DNA polymerase ofclaim 2, further comprising the sequence of SEQ ID NO:5 joined to theamino terminus thereof.
 6. The engineered DNA polymerase of claim 2,further comprising the sequence of SEQ ID NO:6 joined to the aminoterminus thereof.
 7. The engineered DNA polymerase of claim 1, whereinthe substitution mutation at position K250 comprises a mutation to apolar amino acid, wherein the substitution mutation at position Q281comprises a mutation to an acidic amino acid, wherein the substitutionmutation at position D355 comprises a mutation to a different acidicamino acid, wherein the substitution mutation at position Q425 comprisesa mutation to a different polar amino acid, and wherein the substitutionmutation at position D532 comprises a mutation to a different acidicamino acid.
 8. The engineered DNA polymerase of claim 7, wherein thesubstitution mutation at position K250 comprises a mutation to Cys,wherein the substitution mutation at position Q281 comprises a mutationto Glu, wherein the substitution mutation at position D355 comprises amutation to Glu, wherein the substitution mutation at position Q425comprises a mutation to Cys, and wherein the substitution mutation atposition D532 comprises a mutation to Glu.
 9. The engineered DNApolymerase of claim 1, wherein said variant comprises replacement of upto 10 amino acids of SEQ ID NO:3.
 10. The engineered DNA polymerase ofclaim 9, wherein said variant comprises replacement of up to 5 aminoacids of SEQ ID NO:3.
 11. The engineered DNA polymerase of claim 1,wherein said variant is present in a ternary complex that furtherincludes a primed template nucleic acid and a cognate nucleotide oranalog thereof.
 12. The engineered DNA polymerase of claim 11, whereinthe cognate nucleotide or analog thereof comprises an exogenousfluorescent label.
 13. The engineered DNA polymerase of claim 1, whereinthe at least one amino acid substitution mutation is a substitutionmutation at position Q281 that replaces Gln (Q) with Glu (E).
 14. Theengineered DNA polymerase of claim 1, wherein the at least one aminoacid substitution mutation is a substitution mutation at position K250that replaces Lys (K) with Cys (C), and a substitution mutation atposition Q425 that replaces Gln (Q) with Cys (C).
 15. The engineered DNApolymerase of claim 1, wherein the at least one amino acid substitutionmutation is a substitution mutation at position Q281 that replaces Gln(Q) with Glu (E), a substitution mutation at position K250 that replacesLys (K) with Cys (C), and a substitution mutation at position Q425 thatreplaces Gln (Q) with Cys (C).
 16. The engineered DNA polymerase ofclaim 1, wherein the at least one amino acid substitution mutation is asubstitution mutation at position D355 that replaces Asp (D) with Glu(E), and a substitution mutation at position Q281 that replaces Gln (Q)with Glu (E).
 17. The engineered DNA polymerase of claim 1, wherein theat least one amino acid substitution mutation is a substitution mutationat position D355 that replaces Asp (D) with Glu (E), a substitutionmutation at position K250 that replaces Lys (K) with Cys (C), and asubstitution mutation at position Q425 that replaces Gln (Q) with Cys(C).
 18. The engineered DNA polymerase of claim 1, wherein the at leastone amino acid substitution mutation is a substitution mutation atposition D355 that replaces Asp (D) with Glu (E), a substitutionmutation at position Q281 that replaces Gln (Q) with Glu (E), asubstitution mutation at position K250 that replaces Lys (K) with Cys(C), and a substitution mutation at position Q425 that replaces Gln (Q)with Cys (C).
 19. The engineered DNA polymerase of claim 1, furthercomprising an exogenous label covalently joined thereto.
 20. Theengineered DNA polymerase of claim 19, wherein the exogenous labelcomprises a fluorescent label.
 21. The engineered DNA polymerase ofclaim 1, wherein the engineered DNA polymerase comprises Mg²⁺-dependentphosphodiester bond forming activity.
 22. The engineered DNA polymeraseof claim 1, wherein the differential affinity of the engineered DNApolymerase for the primed template nucleic acid in the presence andabsence of cognate nucleotide is greater than the differential affinityof the DNA polymerase of SEQ ID NO:4 for the primed template nucleicacid in the presence and absence of cognate nucleotide.
 23. An isolatedmutant DNA polymerase comprising a variant of the sequence of SEQ IDNO:2, said variant being at least 80% identical to SEQ ID NO:2 andwherein the variant comprises Glu (E) at position
 290. 24. An isolatedmutant DNA polymerase comprising a variant of the sequence of SEQ IDNO:2, said variant being at least 80% identical to SEQ ID NO:2 andwherein the variant comprises Cys (C) at position 259, and Cys (C) atposition
 434. 25. An isolated mutant DNA polymerase comprising a variantof the sequence of SEQ ID NO:2, said variant being at least 80%identical to SEQ ID NO:2 and wherein the variant comprises Glu (E) atposition 290, Cys (C) at position 259, and Cys (C) at position
 434. 26.An isolated mutant DNA polymerase comprising a variant of the sequenceof SEQ ID NO:2, said variant being at least 80% identical to SEQ ID NO:2and wherein the variant comprises Glu (E) at position 364, and furthercomprises Glu (E) at position
 290. 27. An isolated mutant DNA polymerasecomprising a variant of the sequence of SEQ ID NO:2, said variant beingat least 80% identical to SEQ ID NO:2 and wherein the variant comprisesGlu (E) at position 364, and further comprises Cys (C) at position 259and Cys (C) at position
 434. 28. An isolated mutant DNA polymerasecomprising a variant of the sequence of SEQ ID NO:2, said variant beingat least 80% identical to SEQ ID NO:2 and wherein the variant comprisesGlu (E) at position 364, and further comprises Glu (E) at position 290,Cys (C) at position 259, and Cys (C) at position
 434. 29. A reactionmixture, comprising: a DNA polymerase selected from the group consistingof, (i) an engineered DNA polymerase that comprises a variant of thesequence of SEQ ID NO:3, said variant being at least 80% identical toSEQ ID NO:3 and comprising an amino acid substitution mutation at one ormore of positions K250, Q281, D355, Q425, and D532, (ii) an engineeredDNA polymerase that comprises a variant of the sequence of SEQ ID NO:2,said variant being at least 80% identical to SEQ ID NO:2 and wherein thevariant comprises Glu (E) at position 290, (iii) an engineered DNApolymerase that comprises a variant of the sequence of SEQ ID NO:2, saidvariant being at least 80% identical to SEQ ID NO:2 and wherein thevariant comprises Cys (C) at position 259, and Cys (C) at position 434,and (iv) an engineered DNA polymerase that comprises a variant of thesequence of SEQ ID NO:2, said variant being at least 80% identical toSEQ ID NO:2 and wherein the variant comprises Glu (E) at position 290,Cys (C) at position 259, and Cys (C) at position 434; a primed templatenucleic acid molecule, optionally comprising a reversible terminatornucleotide at a 3′-end thereof; and at least one nucleotide.
 30. A kitfor identifying the cognate nucleotide for a primed template nucleicacid molecule, comprising: a DNA polymerase selected from the groupconsisting of, (i) an engineered DNA polymerase that comprises a variantof the sequence of SEQ ID NO:3, said variant being at least 80%identical to SEQ ID NO:3 and comprising an amino acid substitutionmutation at one or more of positions K250, Q281, D355, Q425, and D532,(ii) an engineered DNA polymerase that comprises a variant of thesequence of SEQ ID NO:2, said variant being at least 80% identical toSEQ ID NO:2 and wherein the variant comprises Glu (E) at position 290,(iii) an engineered DNA polymerase that comprises a variant of thesequence of SEQ ID NO:2, said variant being at least 80% identical toSEQ ID NO:2 and wherein the variant comprises Cys (C) at position 259,and Cys (C) at position 434, and (iv) an engineered DNA polymerase thatcomprises a variant of the sequence of SEQ ID NO:2, said variant beingat least 80% identical to SEQ ID NO:2 and wherein the variant comprisesGlu (E) at position 290, Cys (C) at position 259, and Cys (C) atposition 434; a plurality of nucleotides or analogs thereof; and aplurality of reversible terminator nucleotides.
 31. A method ofdetermining whether a test nucleotide is the next correct nucleotidecomprising a base complementary to the next base in a template strandimmediately downstream of a primer in a primed template nucleic acid,comprising the steps of: (a) contacting the primed template nucleic acidwith a first reaction mixture that comprises a crippled DNA polymeraseand the test nucleotide, whereby, if the test nucleotide is the nextcorrect nucleotide, there is formed a complex comprising the primedtemplate nucleic acid, the crippled DNA polymerase and the testnucleotide, wherein the crippled DNA polymerase is substantially unableto catalyze formation of phosphodiester bonds in the presence ofmagnesium ions, and wherein the primer of the primed template nucleicacid comprises a reversible terminator nucleotide at its 3′-end; (b)measuring binding of the primed template nucleic acid to the crippledDNA polymerase in the presence of the test nucleotide, without chemicalincorporation of the test nucleotide into the primer of the primedtemplate nucleic acid; and (c) determining from the results of step (b)whether the test nucleotide is the next correct nucleotide.